A series of pyrimido[4,5-b]quinolin-2,4(1H,3H) dione derivatives (5-deazaalloxazines) was synthesized by the reaction of various substituted 2-chloroquinolin-3-carboxylic acids with urea/thiourea. The structure of these newly synthesized compounds was characterized by standard spectroscopic and analytical techniques. The antimicrobial activities of the title compounds were evaluated against Gram-positive and Gram-negative bacteria and fungi using a micro dilution procedure and compared with those of standard drugs. The minimum inhibitory concentration of the derivative compounds was also determined by measuring their in vitro activities against both bacteria and fungi strains.