ESKAPE pathogens are considered as global threats to human health. The discovery of new molecules for which these pathogens have not yet developed resistance is a high medical priority. Synthetic flavonoids are good candidates for developing new antimicrobials. Therefore, we report here the potent in vitro antibacterial activity of BrCl-flav, a representative of a new class of synthetic tricyclic flavonoids. Minimum inhibitory/bactericidal concentration, time kill and biofilm formation assays were employed to evaluate the antibacterial potential of BrCl-flav. The mechanism of action was investigated using fluorescence and scanning electron microscopy. A checkerboard assay was used to study the effect of the tested compound in combination with antibiotics. Our results showed that BrCl-flav displayed important inhibitory activity against all tested clinical isolates, with MICs ranging between 0.24 and 125 µg/mL. A total kill effect was recorded after only 1 h of exposing Enterococcus faecium cells to BrCl-flav. Additionally, BrCl-flav displayed important biofilm disruption potential against Acinetobacter baumannii. Those effects were induced by membrane integrity damage. BrCl-flav expressed synergistic activity in combination with penicillin against a MRSA strain. Based on the potent antibacterial activity, low cytotoxicity and pro-inflammatory effect, BrCl-flav has good potential for developing new effective drugs against ESKAPE pathogens.