1,8-Diaminonaphthalene was used as an active binucleophile from which some perimidines and (naphthalene-1,8-diyl)bis(heterocycles) were designed, prepared, and assessed for their antimicrobial activity against Staphylococcus aureus, Escherichia coli, different strains of methicillin-resistant Staphylococcus aureus (MRSA), and multidrug resistant (MDR) clinical isolates of Enterococcus faecium and Enterococcus faecalis. Compounds 1 and 6 exhibited significant antibacterial activity against Staphylococcus aureus with MIC values of 62.5 and 15.63 µg/mL. Additionally, compound 2 showed a remarkable antimicrobial activity against MRSA 1 (MIC = 62.5 µg/mL). Additionally, compound 6 exerted extremely potent antimicrobial effect towards all resistant strains with MIC values range of 31.25-62.5 µg/mL that is much more potent than reference drugs amoxicillin and cephalexin (MIC > 500 µg/mL). Furthermore, compounds 2, 3, and 6 displayed powerful antibiofilm activity against both Staphylococcus aureus and MRSA where compound 2 presented the most potent antibiofilm action among the tested compounds. To investigate the mechanism of action for compounds 2 and 6, inhibition of DNA gyrase together with topoisomerase IV were evaluated. Both compounds presented promising inhibitory action with IC50 at the micromolar range.