Junior Zindoga scored the only goal of the game in the 18th minute at the Seisa Ramabodu Stadium.
Usuthu appeared to have equalised in the second half but he goal wasn't given as the ball was adjudged to have not crossed the line.
Vilakazi's side then had a penalty appeal waved away after Rowan Human was upended inside the box.
Speaking after the game, the coach said on SuperSport: "Firstly, I'm very disappointed with the referee today.
"Everything was going against us, but we are very proud of Arthur [Zwane] for the effort of the players. They were working very hard, it was just not meant to be, but the effort was there."
Vilakazi added: "You can check maybe their goal, maybe that was not our best structures defensively and that's why we suffered, but other than that, they worked very hard and created chances which needed to be converted.
"I'm very much disappointed in the referees and their decisions apart from the goal, we are not a team that always complains about referees but this game was very bad and we suffered. That's disappointing for our football in South Africa."
Orlando Pirates, Kaizer Chiefs and Mamelodi Sundowns may have avoided each other in the Carling Knockout round of 16, but their respective fixtures present different challenges as they begin their campaigns in the competition.
AmaZulu claimed local bragging rights with a 2-1 victory over Durban City in Tuesday night's Betway Premiership derby at Chatsworth Stadium, handing Eric Tinkler a disappointing start to his tenure as Citizens coach.
AmaZulu will be targeting a second KwaZulu-Natal derby victory when they face Durban City in the Betway Premiership at Chatsworth Stadium on Tuesday evening.
Mamelodi Sundowns announced the signing of AmaZulu centre-back Wandile Dube as the transfer window drew to a close.
