The purpose of the present study was to evaluate the antimicrobial effects of 3-hetarylazoquinoline-2,4-diols (19), having substituted or unsubstituted thiazole, benzothiazole, benzimidazole, triazole, isoxazole, and thiadiazole moieties at the 3-position, on bacterial and yeast strains. In addition, compounds and DNA interaction with pUC18 plasmid DNA were studied. The biological effects of these molecules were compared with potential antibiotics ampicillin, chloramfenicol (for bacteria), and ketoconozole (for yeast). According to the results, all compounds except 8 have mild to moderate activity against some of the bacterial strains tested, and none of the compounds have activity against the fungi C. tropicalis and C. albicans. Compounds 1 and 6 have moderate activities against B. cereus and B. subtilis, and compounds 5 and 6 have moderate activities and 1 has mild activity against E. coli. Compound 2 has a mild effect on all bacterial strains except E. coli ATCC 25922, P. vulgaris, and S. aureus. Compounds 3-5 and 7 have mild activity against some of the bacterial strains tested. Compound 9 has mild activity against all bacterial strains except E. coli ATCC 25922. Compound 8 has no antibacterial effect. Compounds and DNA interaction results indicate that as the compounds bind to DNA, unwinding of supercoiled form I DNA takes place to change it from the negatively supercoiled form I to the nicked form II and linear form III DNA. The following studies showed that, among the synthesized compounds, 3-9 seem to be promising candidates possessing DNA cleavage activities besides anti-microbial properties to serve as chemical nucleases and chemotherapeutic agents.