A series of new compounds characterized by presence of quinazoline scaffold and phosphorothioate moiety in their molecular structure, was prepared through reacting Japanese reagent (JR, 1a) and Lawesson reagent (LR, 1b) with quinazoline-4-ones (3a-e) in boiling toluene. The expected quinazoline-4-thiones were also formed and well identified. Molecular docking studies were performed to determine the molecular affinity between the new products and the target protein. The starting quinazolines and ten of the new products were in vitro evaluated as antimicrobial agents using Cephradine and Fluconazole as reference drugs for antibacterial and antifungal assays, respectively. Of particularly, the dioxathiaphosphinane (12) and benzoxaphospholylidene (17) exhibited 15% potent inhibition that equals to Cephradine against Escherichia coli strains.