Footage, which came to light on Wednesday, appears to show him with a sweet, or mint, in his mouth and rubbing saliva into the ball, with the International Cricket Council alleging he broke their code of conduct.
“The alleged incident happened on Tuesday morning when TV footage appeared to show du Plessis applying saliva and residue from a mint or sweet, an artificial substance, to the ball in an attempt to change its condition,” it said.
Du Plessis, the acting skipper with AB de Villiers out injured, pleaded not guilty.
A hearing will now be held by Andy Pycroft of the ICC match referees panel. The results will be announced in due course, the ICC statement said.
The South African squad appeared before media in Melbourne on Friday to defend him.
“The reason everyone is here is to stand together, really, and to show solidarity to something… we thought was actually a joke,” veteran batsman Hashim Amla told reporters.
Under cricket rules, a player may shine the ball provided no artificial substance is used with du Plessis potentially facing a fine of up to 100 percent of his match fee.
South Africa easily won the Hobart Test by an innings and 80 runs, plunging Australian cricket into crisis.
It was a fifth straight defeat for Australia, which saw chairman of selectors Rod Marsh quit and Trevor Hohns temporarily replace him.
The third and final Test starts on November 24 in Adelaide.