The footage from December showed a dark stain spread down the 71-year-old president's grey trousers as he stood for the national anthem at a road commissioning event.
The video never aired on television but subsequently circulated on social media.
The journalists, who work with the state-run South Sudan Broadcasting Corporation, were detained on Tuesday and Wednesday, according to Patrick Oyet, president of the South Sudan Union of Journalists.
They "are suspected of having knowledge on how the video of the president urinating himself came out," he told Reuters.
South Sudan Information Minister Michael Makuei and National Security Service spokesman David Kumuri did not immediately respond to requests for comment.
Kiir has been president since South Sudan gained independence in 2011.
Government officials have repeatedly denied rumours circulating on social media that he is unwell.
The country has been embroiled in conflict for much of the past decade.
The detained journalists are camera operators Joseph Oliver and Mustafa Osman; video editor Victor Lado; contributor Jacob Benjamin; and Cherbek Ruben and Joval Toombe from the control room, Oyet said.
"We are concerned because those who are detained now have stayed longer than what the law says," he added.
By law, South Sudanese authorities are allowed to detain suspects for only 24 hours before bringing them before a judge.
The incident "matches a pattern of security personnel resorting to arbitrary detention whenever officials deem coverage unfavourable," the sub-Saharan Africa representative for the Committee to Protect Journalists, Muthoki Mumo, said.
