Series of thiophene, thiazole, and pyrimidine derivatives based on the ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate starting material were synthesized, and cytotoxic activity of synthesized compounds was evaluated. Results showed that three compounds, viz. (2)-2-(4-methyl-3-phenylthiazole-2(3H)-ylidenea-mino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile, ethyl 2-(6-amino-4-imino-3-phenyl-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate, and 4-imino-3-phenyl-3,4,5,6,7,8-hexahydrobenzo[4,5]-thieno[2,3-d]pyrimidine-2-thiol, were the most active towards MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer), but they were not active towards normal fibroblasts human cell line (WI-38). The toxicity of selected compounds against shrimp larvae was also studied.