Amnesty International on Thursday accused Nigeria's security forces of killing at least 150 pro-Biafra protesters and injuring hundreds since August 2015.
The army denied the charge.
"This deadly repression of pro-Biafra activists is further stoking tensions in the southeast of Nigeria," Amnesty warned in its report.
"This reckless and trigger-happy approach to crowd control has caused at least 150 deaths and we fear the actual total might be far higher," said Makmid Kamara, interim director of Amnesty International Nigeria.
The watchdog said its report was based on analysis of 87 videos, 122 photographs and 146 eyewitness accounts relating to demonstrations and other gatherings of Biafran activists between August 2015 and August 2016.
The protesters were asking for a separate state for the Igbo people of southeast Nigeria.
Amnesty said the military used live ammunition with little or no warning to disperse the protesters.
In May 2016, it said, at least 60 people were shot dead and 70 injured in two days of events to mark Biafra Remembrance Day.
Amnesty called for an inquiry.
"The Nigerian government?s decision to send in the military to respond to pro-Biafra events seems to be in large part to blame for this excessive bloodshed," it said.
"The authorities must immediately launch an impartial investigation and bring the perpetrators to book."
Nigerian army spokesman Sani Usman dismissed the allegation.
"We wish to debunk the insinuation that our troops perpetrated the killing of defenceless agitators. This is an outright attempt to tarnish the reputation of the security forces," Usman said in a statement.
He said the activists had in fact killed five policeman at a protest in May and attacked military and police vehicles.
"(...) The military and other security agencies exercised maximum restraints despite the flurry of provocative and unjustifiable violence," he added.
The Indigenous People of Biafra (IPOB) has staged series of demonstrations across the southeast since the arrest in October last year of its leader, Nnamdi Kanu, who has been charged with treason and still in detention.
Kanu, who is also head of the banned Radio Biafra, is accused of calling for a separate republic of Biafra, nearly 50 years after a previous declaration of independence sparked a civil war.
The 1967-70 conflict left more than a million people dead, most of them from starvation and disease, as the Igbo nation was blockaded into submission.
Amnesty and other human rights groups have previously highlighted similar claims against the military in December last year, when at least 350 Shiite Muslim protesters were killed in the northern city of Zaria.
The military has also been accused of a catalogue of abuses against civilians during Boko Haram's Islamist insurgency in the northeast.