The 26-year-old former Rahimo FC centre-back joins Babina Noko after a strong season with Asante Kotoko, playing 34 games in total and scoring six goals.
Sekhukhune made a number of new signings this season but have already dispensed with head coach Cedric Kaze and have announced Papi Zothwane as interim boss.
The likes of Thato Khiba, Lesiba Nku, Nyiko Mobbie, Miguel Timm and Ronaldo Maarman are all part of a Sekhukhune side sitting in sixth place on the league standings with seven points from their opening five games.
"Sekhukhune United is delighted to welcome defender Ndongani Samba Gilbani O’Neil — popularly known as Samba O'Neil to Babina Noko," wrote the club.
"A former Captain of Asante Kotoko in Ghana, Samba brings valuable experience, leadership and defensive authority to the team.
"Welcome to the family, Samba!"
Bradley Grobler scored a late equaliser as Stellenbosch FC played to a 1-1 home draw against Sekhukhune United in their Betway Premiership clash at Athlone Stadium on Friday.
Papi Zothwane will be hoping to guide Sekhukhune United to their first victory since taking over as interim head coach when they face Stellenbosch at Athlone Stadium on Friday.
Sekhukhune United interim coach Papi Zothwane has explained Bright Ndlovu's absence from Saturday's goalless draw with TS Galaxy.
Two Betway Premiership clashes on Saturday produced no goals as Chippa United were held to a goalless draw by newly-promoted Milford, while Sekhuhune and TS Galaxy could also not find the back of the net.
