With the aim of developing potent analgesic, anti-inflammatory, and antimicrobial agents a series of novel quinazolin-4(3H)-one derivatives were synthesized and characterized by FT-IR, 1 H-NMR, mass spectroscopy and bases of elemental analysis. Tail-flick technique, carrageenan-induced foot paw edema test, and agar streak dilution test were performed for screening analgesic, anti-inflammatory, and in vitro antimicrobial activity, respectively. Moreover, all compounds were examined for its ulcerogenicity. Results revealed that entire series of compounds exhibited mild to good analgesic, anti-inflammatory, and antimicrobial activity with low to moderate ulcer index. The relationship between the functional group variation and the biological activity of the evaluated compounds were discussed. Compound 2-(2-(4-(trifluoromethyl)benzylidene)hydrazinyl)-N-(4-(2-methyl-4-oxoquinazolin-3 (4H)-yl) phenyl) acetamide 5e was determined to be the most active compound.