A grand jury in Louisiana indicted Derrick Stafford and Norris Greenhouse Jr on second-degree murder charges over the death of Jeremy Mardis.
The two men opened fire on the car during a traffic stop in Marksville, Louisiana, after a police chase.
The charges come as a national debate rages over police use of deadly force.
It was not clear why the police were in pursuit of Jeremy's father, Chris Few, who was not armed.
The indictment, delivered on Thursday by a grand jury, charges Mr Stafford, 32, and Mr Greenhouse Jr, 23, with one count each of second-degree murder and attempted second-degree murder.
Police have said the two men fired 18 rounds into the car, severely injuring Mr Few and killing his son.
Another police officer who was present when it happened told investigators he had not fired his own weapon because "he didn't fear for his life", according to court documents.
Two US police officers have been charged with the murder of a six-year-old boy who was shot and killed while travelling in his father's car.