AbstractSerious bacterial infections could be caused by Gram‐positive microorganisms, in particular methicillin‐resistant Staphylococcus aureus and Staphylococcus epidermidis. Aiming to address this challenging issue by developing the potent and selective antimicrobial lead structures against methicillin‐resistant Staphylococcus spp., herein, we report in vitro evaluation of quinolinequinones (QQ1–QQ10) against the Gram‐negative and Gram‐positive strains using the broth microdilution technique. The design principle of the quinolinequinones was based on the variation of the structures attached to the 1,4‐quinone moiety and substituent(s) within amino phenyl moiety. A series of ten quinolinequinones displayed activity mainly against the Gram‐positive strains with a minimal inhibitory concentration (MIC=1.22‐1250 mg/L) within the Clinical and Laboratory Standards Institute (CLSI) levels. Interestingly, QQ3, QQ5, and QQ6 displayed equal antibacterial inhibitory activity against S. aureus (MIC=1.22 mg/L), respectively, to the standard positive control Cefuroxime‐Na. QQ2, QQ3, and QQ5 had the best inhibitory activity with the MIC value of 1.22 mg/L (4‐fold more potent compared reference standard Cefuroxime) against S. epidermidis. On the other hand, QQ3 was the most effective quinolinequinone against fungi, in particular C. albicans. The identified lead quinolinequinones (QQ3 and QQ5) with a comprehensive analysis of structure–activity relationships and further studies showed high activity against methicillin‐resistant Staphylococcus spp. It is worth noting that the isopropyl group has importance for excellent bioactivity. Remarkably, the in vitro antibiofilm and bactericidal activities (each of 32 clinically obtained strains of Gram‐positive bacteria) of the selected two quinolinequinones (QQ3 and QQ5) have been evaluated for the mode of action in addition to the time‐kill curve study.