Abstract Guanylhydrazones have shown promising antitumor activity in preclinical tumor models in several studies. In this study, we aimed at evaluating the cytotoxic effect of a series of synthetic guanylhydrazones. Different human tumor cell lines, by including HCT-8 (colon carcinoma), MDA-MB-435 (melanoma) and SF-295 (glioblastoma) were continuous exposed to guanylhydrazone derivatives for 72 hours and growth inhibition of tumor cell lines and macrophages J774 was measured using tetrazolium salt (MTT) assay. Compounds 7, 11, 16 and 17 showed strong cytotoxic activity with IC ₅₀ values lower than 10 μmol L⁻¹ against four tumor cell lines. Among them, 7 was less toxic to non-tumor cells. Finally, obtained data suggest that guanylhydrazones may be regarded as potential lead compounds for the design of novel anticancer agents.