Background: Pyrimidines have shown numerous biological activities such as antimicrobial, anticancer, anticonvulsant, antiviral and anti-inflammatory.

Objective: Encouraged by these remarks, the synthesis of 2-((1H-benzo[d]imidazol-2- yl)methylthio)-4- amino-6-phenylpyrimidine-5-carbonitrile (3a-g) were performed.

Methods: 4-Amino-2-mercapto-6-phenylpyrimidine-5-carbonitrile was dissolved in aqueous sodium hydroxide solution and to this clear solution, 2-Chloromethyl-1H-benzimidazole in methanol was added and the reaction mixture was stirred under reflux to get desired product. The structures of newly synthesized compounds were confirmed by their physical, chemical and spectral data. The synthesized derivatives were screened for their in vitro, antibacterial activity against Gram- positive bacteria: Staphylococcus aureus and Bacillus subtilis and Gram- negative bacteria: Escherichia coli and Pseudomonas aeruginosa by using Ciprofloxacin as reference standard. While their antifungal activity was evaluated against Aspergillus niger and Candida albicans using Fluconazole as reference drug. The docking study was performed to check the interactions of target compounds. All the synthesized compounds (3a-g) were docked with Homo sapiens DHFR (PDB: 1S3V), Bacterial (S aureus) DHFR (PDB: 2W9T) and DHPS (PDB: 1AD4) protein. The dock score and binding interactions were recorded.

Result: Antimicrobial activity indicates that compound 3b, 3f and 3g have showed good activity.The docking study revealed that the compounds 3b, 3f and 3g showed good dock score and comparable interactions compared to reference ligand (Trimethoprim/ Sulfadiazine) which confirms the selectivity.

Conclusion: Thus, we conclude presume that the synthesized compounds have capability for further promotion as novel antimicrobial agents.