Due to the variability and ability of tumor to mutate, as well as the heterogeneity of tumor tissue, such drugs are sought that would act selectively and multidirectionally on the cancer cell. Therefore, two newly synthesized semicarbazide structured substances were evaluated for anticancer properties in our study: 1a and 1b. In order to evaluate the cytotoxicity and selectivity of the tested compounds, MTT and Neutral Red uptake assay on cell lines (HEK293, LN229, 769-P, HepG2 and NCI-H1563) and cell cycle analysis were performed. Acute toxicity and cardiotoxicity were also evaluated in the zebrafish model. The tested compounds (1a, 1b) showed cytotoxic activity, with the greatest selectivity noted against the glioblastoma multiforme cell line (LN229). However, compound 1b showed stronger selective activity than 1a. Both of compounds were shown to significantly affect the M phase of the cell cycle. Whereas, the conducted toxicological examination of newly synthesized thiosemicarbazide derivates showed, that direct exposition of Danio rerio embryos to compound 1a, but not 1b, causes a concentration-dependent increase in developmental malformations, indicating possible teratogenic effects.