Considering that some thiopyrimidines were previously reported as potential therapeutics, the present study achieved novel analogs of bioactive 2-substituted thiopyrimidines-4-(3H)-ones via base catalyzed alkylation reaction of 2-thiouracil using alkyl and aralkyl bromides. The title compounds were 2-(1-butylthio)pyrimidine-4(3H)-one (5a), 2-(2-butylthio)pyrimidine-4(3H)-one (5b), 2-(cyclohexylmethylthio)pyrimidine-4(3H)-one (5c), 2-(benzylthio)pyrimidine-4(3H)-one (5d) and 2-(1-adamantylthio)pyrimidine-4(3H)-one (5e). Bioactivity tests revealed that thiopyrimidines 5a, 5c, 5d and 5e exhibited antimicrobial activity. The thiopyrimidine-4-one (5c) showed complete inhibition against Streptococcus pyogenes and Branhamella catarrhalis as well as antifungal action against Candida albicans. Significantly, the 1-adamantylthiopyrimidine (5e) was shown to be the most potent cytotoxic compound against multidrug-resistant small cell lung cancer (H69AR). Their structure-activity relationships were discussed.