The aim of this research was to examine cytotoxic effects of certain isothiocyanates from the Lepidium family of plants on two cell lines of human carcinomas, UM-UC-3 and LN229. We used distillate and extract of Lepidium graminifolium and Lepidium latifolium and allyl isothiocynate, phenyl isothiocyanate and benzyl isothiocyanate. Both cell lines were treated with concentrations of 1 μg/mL, 5 μg/mL, 10 μg/mL, 50 μg/mL and 100 μg/mL. Cytotoxicity was measured after the periods of 4, 24, 48 and 72 hours, using the MTT assay. The ratio of absorbances of cells treated with isothiocyanates in aqueous solutions and the ones not treated showed the cytotoxic activity of used isothiocyanates. In cell line UM-UC-3, the most significant cytotoxic effect was achieved by benzyl isothiocyanate at the concentrations of 50 μg/mL and 100 μg/mL after 24-hour incubation time. The most prominent effect on the reduction of the number of metabolically active cells in cell line LN229 was achieved by benzyl isothiocyanate at the concentrations of 50 μg/mL and 100 μg/mL after 48-hour incubation time. Tested isothiocyanates showed cytotoxic effects dependent on incubation time and concentration. The effectiveness of isothiocyanates was not always correspondent to the increase of concentration and incubation time and, in some cases, cell recovery occurred. The cytotoxic effect of isothiocyanates is confirmed, which was the hypothesis of the research, and the next step is the analysis of these effects by in vivo studies on the models of animal urinary bladder and glioblastoma cancers.