Carlington Nyadombo scored the decisive goal of the match in the first half.
Santos caused problems down the left flank in the opening stages and in the eighth minute Vuyisile Wana delivered a cross to find Tyren Arendse unmarked at the far post, but his shot was blocked by Marc van Heerden.
The visitors threatened again a minute later when Wana turned inside the box before setting the ball up for Matthew Pattison on the edge of the penalty area, but his first time shot flew over the target.
AmaZulu then started to see more of the ball, but struggled to get behind the Santos defence.
Santos came close to taking the lead five minutes later when the ball fell for defender Nasief Morris inside the box, but his tame effort was palmed away by Tapuwa Kapini.
Stanton Lewis continued to collect the ball from deep in the midfield and in the 32nd minute his lobbed pass picked out Tsweu Mokoro in the Santos penalty area, but his header was pushed wide by Pa Dembo Touray.
Usuthu took the lead a minute later when Nyadombo got in ahead of his marker in the centre of the box to nod home from a Lewis corner to give his side the advantage at the break.
Santos went in search of the equaliser and in the 54th minute Tyren Arendse created some space on the right before firing a low shot which was well-blocked by Kapini.
A minute later substitute Ayanda Dlamini almost extended the lead for the home side with a curled effort that looked to be heading for the top left corner, but in the end the ball sailed just wide of the target.
Former AmaZulu player Pere Ariweriyai almost leveled matters in the 66th minute when he out-jumped everyone in the box to connect with a free-kick from Songwiqi, but Kapini went down quickly to stop the ball on the line.
Usuthu had a number of chances to seal the game in the dying minutes but Siyabonga Vilane headed over the bar from close range in the 84th minute, while Goodman Dlamini sent his shot wide two minutes later.
In the second minute of added time Ayanda Dlamini collected the ball on the right and beat two defenders as he burst into the box, but in the end his low shot lacked pace and Touray was able to make the save.
A minute later Siyabulela Songwiqi had the last chance to net the equaliser when he picked up a loose ball just outside the box, but his powerful strike went wide of the post.
The last chance of the match fell for Goodman Dlamini as he received a pass from Mokoro on the edge of the box, but he shot straight at Touray as Usuthu held on for the narrow win.