Background:Microbial resistance has become a worldwide public health problem, and may lead to morbidity and mortality in affected patients.Objective:Therefore, this work aimed to evaluate the antibacterial activity of quinone-4-oxoquinoline derivatives.Method:These derivatives were evaluated against Gram-positive and Gram-negative bacteria by their antibacterial activity, anti-biofilm, and hemolytic activities and by in silico assays.Results:The quinone-4-oxoquinoline derivatives presented broad-spectrum antibacterial activities, and in some cases were more active than commercially available reference drugs. These compounds also inhibited bacterial adhesion and the assays revealed seven non-hemolytic derivatives. The derivatives seem to cause damage to the bacterial cell membrane and those containing the carboxyl group at the C-3 position of the 4-quinolonic nucleus were more active than those containing a carboxyethyl group.Conclusion:The isoquinoline-5,8-dione nucleus also favored antimicrobial activity. The study showed that the target of the derivatives must be a non-conventional hydrophobic allosteric binding pocket on the DNA gyrase enzyme.