Members of Mr Nichols family were present in the courtroom along with their legal representative, civil rights lawyer Ben Crump.
The ex-officers, dressed in suits and wearing black face masks, stood silently beside their legal representatives.
None spoke during the short hearing. Their lawyers confirmed the not guilty pleas when asked by the judge.
The officers were arrested and taken into custody on 26 January after the Memphis police reviewed bodycam footage of the 29-year-old’s violent arrest.
In the footage, Mr Nichols can be heard calling for his mother as he is beaten by police after being pulled over for alleged reckless driving.
He was pepper sprayed, kicked and punched by the officers and died in hospital three days later.
Memphis Police Chief Cerelyn Davis said the incident was “not just a professional failing,” but “a failing of basic humanity toward another individual”.
Mr Nichols’ mother, RowVaughn Wells, previously told BBC News that it was the race of the victim – in this case her son – and not the race of the perpetrators that mattered.
“It’s not about the colour of the police officer. We don’t care if it’s black, white, pink, purple. What they did was wrong,” she said.
“And what they’re doing to the black communities is wrong. We’re not worried about the race of the police officer. We’re worried about the conduct of the police officers.”