Several new substituted 1,2,3,4-tetrahydropyrimidine derivatives 2a–h have been synthesized by the modified Biginelli and Hantzsch reactions and compounds were characterized by spectral techniques. All compounds were evaluated for their in vitro antimicrobial activity against different strains of Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus and S. pyogenus) bacteria and selected fungi C. albicans, A. niger, and A. clavatus using serial Broth dilution method (Mueller– Hinton broth dilution method). Compound 2e was found active against both Gram-negative and -positive bacterial strains used for present study, while compound 2b was good active against Gram-negative and S. pyogenus bacterial strains. Compounds 2a–h were not that much significant against (C. albicans, A. niger, and A. clavatus) selected fungal strains.