Thousands of protesters felt that the US was not keeping up its commitment to fighting AIDS in Africa and other developed countries.
Protesters wore bright green shirts that said "HIV positive" and carried signs calling on US President Barack Obama to protect access to AIDS treatment as they marched through the posh suburb of Sandton to deliver a letter to the US consulate.
The memo accused the United States of cutting funding to the Global Fund to Fight AIDS, Tuberculosis and Malaria, an international initiative created to combat disease in the developing world.
March organisers said they fear aid cuts by the United States will cause other countries to follow suit and reduce funding for the fight against HIV.
"How does your administration justify cutting funding... at a time when the (Fund) estimates that it needs at least 20 billion dollars from 2011 to 2013 in order to maintain and expand its existing commitments," the letter said.
"Because of the importance of the US commitment to the (fund) in marshalling the commitment of other countries, this proposed decrease stands to have a compounded effect."
The US embassy denied the country was reducing funding for the fight against AIDS, saying Obama's 2011 budget request to Congress included an increase to the president's emergency plan for AIDS relief.
But Sharon Ekambaram of Doctors Without Borders, one of 16 organisations that signed the letter, said doctors and AIDS activists have noticed slackening US support for programmes across Africa.
"There isn't anything in writing, but we are seeing the impact on clinics," she said.
"Projects are being told they can't take on more people. There's not enough money to buy and keep (HIV treatment) drugs in storage," she said.
Ekambaram called on international leaders to use the World Cup, which South Africa is hosting from June 11 to July 11, to focus on the AIDS pandemic as well as football.
"The world's attention is on Africa and South Africa, which is great," she said. "But AIDS doesn't take a rest. It's still devastating people's families and people's lives."