The hosts got off to the perfect start as they grabbed an early goal through Bonginkosi Ntuli, who scored to make it 1-0 to AmaZulu 10 minutes into the game.
City had to find a way to level matters and Kermit Erasmus kept the Usuthu defence busy as he looked to find the back of the net.
The former Orlando Pirates striker was denied by AmaZulu goalkeeper Siyabonga Mbatha just before the half-hour mark.
AmaZulu were leading 1-0 against City during the half-time break.
The visitors piled pressure on Usuthu after the restart with Mark Mayambela looking to impress on his debut for City.
The lanky winger was the man who restored parity as he beat Mbatha to make it 1-1 just after the hour-mark.
However, Ntuli had other ideas in stoppage time as he grabbed his brace to seal AmaZulu's 2-1 victory over City on the night.
AmaZulu's first win of the season took them to 14th spot on the league standings, while City remained 10th on the table.
Meanwhile, Bidvest Wits secured a 1-0 victory over Chippa United in a match which took place at Nelson Mandela Bay Stadium in Port Elizabeth.
The two teams attacked each other as they looked to break the deadlock, but they could not find the back of the net in the first half.
The score was 0-0 at the interval between Chippa and Wits.
Gift Motupa, who scored nine goals in the league last season, was introduced in the second half as Wits looked to open the scoring.
The visitors pushed for a late winning goal and they managed to score with one minute left on the clock.
Deon Hotto set-up Buhle Mkhwanazi, who hit the back of the net to seal Wits' 1-0 victory over Chippa on the night.
The win took Wits to the second spot on the league standings, while Chippa slipped down to 16th place on the table.