A new series of 6- bromo-2-(4-chlorophenyl)quinazolin-4(3H)-one derivatives 2-7 were synthesized. Their chemical structures were confirmed by spectral and elemental analysis. The synthesized compounds were evaluated for their anti-microbial activity using the agar diffusion technique. The tested compounds were evaluated against, Grampositive bacteria (Bacillus subtilis, Staphylococcus aureus), Gram- negative bacteria (Escherichia coli and Pseudomonas aeruginosa ), Yeast (Candida albicans) and Filamentous Fungus (Aspergillus niger ). Minimum inhibitory concentration (MIC) of the compounds which showed antimicrobial activity was determined in vitro. Compound 3b has broad spectrum activity against Gram-positive bacteria, Gram-negative bacteria, Yeast and Filamentous Fungus with MICs 150, 150, 75, 312, 625 and 625 μg/mL against (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 3555, Escherichia coli ATCC 23282 Pseudomonas aeruginosa ATCC 10145, Candida albicans IMRU 3669 and Aspergillus niger ATCC 16404) respectively.