The present work describes the design and development of seventeen pyrimidine-clubbed benzimidazole derivatives as potential dihydrofolate reductase (DHFR) inhibitors. These compounds were filtered by using ADMET, drug-likeness characteristics calculations, and molecular docking experiments. Compounds 27, 29, 30, 33, 37, 38, and 41 were chosen for the synthesis based on the results of the in silico screening. Each of the synthesized compounds was tested for its in vitro antibacterial and antifungal activities using a variety of strains. All the compounds showed antibacterial properties against Gram-positive bacteria (Staphylococcus aureus and Staphylococcus pyogenes) as well as Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Most of the compounds either had a higher potency than chloramphenicol or an equivalent potency to ciprofloxacin. Compounds 29 and 33 were effective against all the bacterial and fungal strains. Finally, the 1,2,3,4-tetrahydropyrimidine-2-thiol derivatives with a 6-chloro-2-(chloromethyl)-1H-benzo[d]imidazole moiety are potent enough to be considered a promising lead for the discovery of an effective antibacterial agent.