Due to the current need to identify novel and potent drug candidates for treating diseases caused by bacterial and fungal strains.Herein, in this article, our goal is to identify possible potential antimicrobial candidates.The present work includes design and prepare three new series of quinazolindione-based acylthiourea incorporating five and/or six membered rings such as pyrazole, furo[2,3-b]indole and chromene moieties 3-8, in an effort to investigate their potential antimicrobial properties.The starting compound 2 has been synthesized by reaction of benzoyl chloride derivative 1 with cyanoacetic acid hydrazide and ammonium thiocyanate via one pot three components reaction.Addnl., Knovenagel condensation of 2 with various heteroaromatic/ aromatic aldehydes and/or aromatic ketone yielded quinazolin-2,4-diones-anchored acylthiourea conjugate with N/O heterocycles 3-8 in good yields ranging from 60 to 75 %.Their chem. structures were authenticated utilizing multispectroscopic techniques such as IR, 1H-& 13C NMR and MS along with the elemental analyses.Further, the antimicrobial potential of the products was in vitro assessed against different bacterial and fungal pathogenic strains.The results revealed that compounds 3b and 3c demonstrated the strongest inhibitory effects against the tested microbial strains at low concentrationsAlongside, virtual screening based on mol. docking studies was further performed on the newly synthesized quinazolin-2,4-diones towards the enzymes Staphylococcus aureus tyrosyl-tRNA synthetase (TyrRS), Pseudomonas aeruginosa lipase enzyme and Candida albicans dihydrofolate reductase (C. albicans DHFR), using ciprofloxacin and nystatin as antibacterial and antifungal standard drugs, resp.Further, structure activity relationship (SAR) for 3b and 3c was rationalized by investigating the effect of substituents on inhibitory potential.A semiempirical modeling was performed for geometric optimization of the best docked mols.Our work recommends that compound 3b and 3c could be promising leads for development of potent antimicrobial agents.