This study synthesized a series of 3-((4-(4-nitrophenyl)-6-aryl-1,6-dihydropyrimidin-2-yl)thio)propanenitriles and their structures characterized by spectral (H-1 NMR, C-13 NMR, infrared spectroscopy, liquid chromatography-mass spectroscopy) and elemental analysis. Antimicrobial activity was carried out by serial broth dilution method on gram-positive bacteria (Staphylococcus aureus and Streptococcus pyogenes), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and three fungi (Candida albicans, Aspergillus niger and Aspergillus clavatus) in vitro. Compounds 5d, 5j, 5l and 5m showed potent antimicrobial activity against tested microorganisms. The grades of cytotoxicity studies on a human cervical cancer cell line (HeLa) and a 3T3 mouse embryonic fibroblast cell line indicated that compounds 5c, 5d, 5h and 5j showed low cytotoxicity.