Zimbabwe's Foreign Affairs Minister Sibusiso Moyo said he had summoned US Ambassador Brian Nichols to express his government's displeasure over Mr O'Brien's statements.
The Minister said the government was reflecting "on the lack of balance and even the double standards so evident in US policy towards Zimbabwe."
The ouster of the late Mugabe in a military coup in 2017 has not led to an improvement in relations with the US.
Zimbabwe on Monday summoned the US Ambassador to Harare to protest a Washington official's description of the southern African country as an adversary amid raging anti-racism protests.
This comes after US national security advisor Robert O'Brien classified Zimbabwe, China and Russia as some of America's adversaries. He claimed that the three countries were trying to take advantage of the recent unrest sparked by the murder of African American George Floyd by a white policeman.
