Despite the victory, Usuthu, who had defeated Mamelodi Sundowns in midweek, remained 11th on the league table, while the Citizens also remained fourth on the table - three points behind third-placed Orlando Pirates.
New signing Talent Chawapiwa was named amongst the substitutes as Usuthu looked to avoid another league defeat to City, who emerged 3-0 winners in the first round clash.
Kermit Erasmus started upfront alongside speedy forwards Craig Martin and Gift Links in a 4-3-3 formation as City adopted an offensive approach against AmaZulu.
The visitors dominated the first 20 minutes of the game with Usuthu struggling to cope with City's speedy front three, who were using their pace to penetrate the hosts' defence.
City were awarded a penalty after Erasmus was fouled inside the AmaZulu box in the fifth minute, but the diminutive striker was superbly denied by goalkeeper Siyabonga Mbatha from the spot-kick.
Mbatha, whose form between the sticks has kept experienced shot-stopper Moeneeb Josephs on the bench, then produced a magnificent save to deny Links from a one-on-one situation.
Usuthu grew in confidence as the match progressed, but they could not seriously test City keeper Peter Leeuwenburgh with Kwanda Mngonyama looking solid at the heart of the visitors' defence.
The score was 0-0 at the interval thanks to some brilliant goalkeeping by Mbatha.
The Citizens looked to continue their dominance after the restart and they came very close to scoring when Teko Modise's effort beat Mbatha, but the crossbar came to Usuthu's rescue.
The hosts were then awarded a free-kick in a promising area following a rare attack by Cavin Johnson's side just after the hour-mark, and Tapelo Nyongo stepped up and beat Leeuwenburgh from the set-piece to make it 1-0 to AmaZulu.
Benni McCarthy had to react and he introduced former Helsingborg FC attacking midfielder Bradley Ralani as the visitors looked to grab a late equalizing goal.
The Citizens, who had netted the majority of their goals (31%) in the final 15 minutes of their league games this season, piled pressure on AmaZulu in the latter stages of the match.
However, Mbatha produced a great low save to deny Thamsanqa Mkhize and ensured that AmaZulu secured a 1-0 win over City, whose six-match unbeaten run in the league came to an end.