Nduduzo Makhathini was born on September 24, 1982, in Pietermaritzburg, the seat of the uMgungundlovu district in KwaZulu-Natal, and grew up in the surrounding hill country, the site of the Zulu king Dingane's kingdom in the 1830s. His mother, Nomajerusalema, gave him his first piano lessons, and his late father, Sibusiso, played guitar and sang in the maskandi style. The family was strictly Christian, and as a boy he walked from church to church to hear the music, often leaving before the sermon. He has described arriving at jazz late and almost by accident: he went to study music in Durban, entered the jazz programme at what was then the Durban Institute of Technology in 2001, and earned a diploma in jazz piano in 2005. At about eighteen he met the pianist Bheki Mseleku, who became his mentor and introduced him to the records of John Coltrane's classic quartet with McCoy Tyner.

After appearances at the National Arts Festival in Grahamstown in 2005 and 2006, he toured Europe in 2006 with the singer Simphiwe Dana and played at the Avo Session festival in Basel. From 2007 he toured for years in saxophonist Zim Ngqawana's Zimology Quartet, which gave him his first recording credit, the 2007 album Live at Bird's Eye, Switzerland, and he is also heard on bassist Herbie Tsoaeli's African Time (2012). In 2014 he and his wife, the singer Omagugu Makhathini, founded the label Gundu Entertainment, and on September 5 of that year he released two albums at once, Mother Tongue and Sketches of Tomorrow. Listening to the Ground and Matunda Ya Kwanza, Vol. 1 followed in 2015, the year he won the Standard Bank Young Artist Award for jazz, and Icilongo: The African Peace Suite in 2016, with Shabaka Hutchings on tenor saxophone. Inner Dimensions, made with his Umgidi Trio and the One Voice Vocal Ensemble, won the South African Music Award for best jazz album in May 2017, and the same year he took the best jazz artist prize at the All Africa Music Awards. He is also on Wisdom of Elders (2016), by Hutchings's group Shabaka and the Ancestors.

His 2017 album Ikhambi, licensed from Gundu to Universal Music South Africa, won the best jazz album award at the South African Music Awards again in 2018, and he went on to become the first South African artist signed to Blue Note Records. By 2017 he was head of the music department at the University of Fort Hare in the Eastern Cape. In 2019 he made his first appearances at the Blue Note club in New York and at Jazz at Lincoln Center, where he was a featured guest of Wynton Marsalis and the Jazz at Lincoln Center Orchestra. His Blue Note debut, Modes of Communication: Letters from the Underworlds (April 3, 2020), set compositions about healing and ancestral worlds for a multi-horn band that included the American altoist Logan Richardson, tenor saxophonist Linda Sikhakhane, trumpeter Ndabo Zulu, bassist Zwelakhe-Duma Bell le Pere and drummer Ayanda Sikade. The New York Times named it one of the best jazz albums of 2020. In the same year he played Fender Rhodes on two tracks of Shabaka and the Ancestors' We Are Sent Here by History.

Makhathini is also a practicing sangoma, a traditional Zulu healer. He has said he first sensed the calling at thirteen and accepted it about a decade later, and his healing practice informs his albums. In the Spirit of Ntu (May 27, 2022), conceived during the 2021 unrest in Durban and the pandemic, was the first release on Blue Note's new imprint Blue Note Africa and used a young South African band: Sikhakhane, trumpeter Robin Fassie Kock, vibraphonist Dylan Tabisher, bassist Stephen de Souza, percussionist Gontse Makhene and drummer Dane Paris, with the American altoist Jaleel Shaw and the singers Omagugu Makhathini and Anna Widauer as guests. In March 2023 he received a PhD in music from Stellenbosch University, and that June he stepped down as head of the Fort Hare music department and moved back to KwaZulu-Natal. The trio album uNomkhubulwane (June 7, 2024), a three-movement suite named for a Zulu deity and drawn from a song that came to him during his healer's initiation, was recorded with bassist Bell le Pere and drummer Francisco Mela. On June 13, 2025, at the Deutscher Jazzpreis ceremony in Cologne, he won the award for international live act of the year. His fourth Blue Note album, The Myth We Choose, appeared on June 26, 2026, co-produced with his son Thingo, with his working trio of bassist Dalisu Ndlazi and drummer Lukmil Perez (and Ayanda Sikade on some tracks) and guests including Hutchings, the producer Black Coffee and Omagugu Makhathini.

Makhathini plays piano in the modal, rhythmically driving manner of McCoy Tyner, a lineage he traces through Mseleku, and blends it with the gospel-tinged melodic warmth of Abdullah Ibrahim and the dance rhythms of Zulu and Afro-Cuban music. He also names Moses Molelekwa, Andrew Hill, Randy Weston and Don Pullen among his influences. Reviewers place his records between spiritual jazz and post-bop: London Jazz News heard in Modes of Communication slow-building solos over repeated rhythm figures in the Coltrane tradition, while uNomkhubulwane strips the sound to a trio and The Myth We Choose adds neo-soul and electronic touches that he credits to his son. He treats a performance as a ritual and an album as a record of messages received from ancestral worlds, writes his own liner notes and essays, and sings on his later albums. His career has run in parallel as an educator and researcher, and his doctorate combined artistic practice with written research. He has gathered a younger generation of South African players around him, among them Linda Sikhakhane, Ayanda Sikade and Zwelakhe-Duma Bell le Pere.