More than 30 people, largely supporters of former president Evo Morales, were killed amid unrest in late 2019 following a vote in which Morales was elected but later resigned and left the country amid accusations of election fraud.
Anez then assumed power, taking office for less than a year.
Attorney General Juan Lanchipa told a press conference that specialised military forces "used weapons with war ammunition against civilians," resulting in deaths and multiple injuries from "the impact of firearm projectiles".
"These events occurred after planning and execution of joint military police operations ordered by the government through the army and police commanders," Lanchipa said.
An accusation was formally presented against Anez and some of her former government colleagues as well as former military and police chiefs, Lanchipa added.
Anez's legal team did not immediately reply to a request for comments on the new charges.
Last year, a Bolivian court found Anez guilty of orchestrating a coup that brought her to power during the 2019 political crisis.
She is in prison after receiving a 10-year sentence.
Anez stated she is innocent of the coup charges, arguing that her ascension to the top office helped calm a tense country and lay the groundwork for elections in October 2020.
