Aaron Torgalski, 39, and Robert McCabe, 32, pleaded not guilty in Buffalo, New York. They were released without bail, and face up to seven years in prison.
On Thursday, they were seen shoving Martin Gugino, who fell backwards on the pavement and started bleeding.
He remains in a local hospital in a serious but stable condition.
The two officers were enforcing a curfew in the city as a result of protests since the death of George Floyd in police custody in Minneapolis last month.
Aaron Torgalski and Robert McCabe, members of the Emergency Response Team, were suspended without pay after footage of the incident outside the City Hall went viral.
Fifty-seven of their colleagues - the entire unit - later resigned from the team in response to the officers' suspension.
On Saturday, a crowd of more than 100 supporters - including police officers and firefighters - protested outside the courthouse in Buffalo against the assault charges filed.
What are Buffalo prosecutors saying?
In a statement, Erie County District Attorney John Flynn said: "The two defendants, who are Buffalo Police officers, pushed a protestor outside of City Hall, causing him to fall and hit his head on the sidewalk."
He stressed that he was not taking sides in the high-profile case.
"We're on the same team here. We're all working each and every day to do justice, to keep our streets safe, to keep our communities safe.
"I'm partnered with law enforcement every day to do that. And when I have to prosecute one of my teammates it doesn't help the situation," Mr Flynn said.