Persistence of skin and wound infections is nowadays accepted being linked to bacterial biofilms, which are highly recalcitrant to treatments and contribute to maintain a constant inflammation state and prevent a correct healing. Topical antimicrobials are the most common first-line self-medications; however, treatment failure is not uncommon and emerging resistance to antibiotics is alarming. Chlorquinaldol is an antimicrobial with a wide spectrum of activity and desirable characteristics for topical application. Aim of this study was to evaluate the efficacy of chlorquinaldol to prevent or eradicate and biofilms, in comparison to classic topical antibiotics like gentamicin and fusidic acid.Minimum inhibitory concentrations (MIC) were assessed for each strain and subinhibitory concentrations (½ and ¼ MIC) were used in the biofilm assay. Antimicrobial assays were performed during biofilm formation or were applied on mature biofilms and were evaluated by means of crystal violet assay and confocal laser scan microscopy.Chlorquinaldol and gentamicin were the most effective antimicrobials in both eradicating and preventing pathogens biofilm; however, resistance to methicillin and impermeability to carbapenems impaired chlorquinaldol effect. In addition, similarly to other hydroxyquinolines, aspecific metal chelation is here proposed as chlorquinaldol mode of action.Relying on an acceptable antibiofilm and a wide spectrum of activity, an aspecific mode of action and consequent absence of resistance development, chlorquinaldol proved to be a good antimicrobial for topical use.