The aim of this research was to examine the cytotoxic effect of samples obtained from Sisymbrium spp. and isopropyl isothiocyanate on three human cancer cell lines: A549, MDA-MB-231 and T24. Distillates and extracts from the plant species Sisymbrium officinale (L.), Sisymbrium orientale and isopropyl isothiocyanate were used. Three 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 determined after 4, 24, 48 and 72 h using the MTT method, but only the most significant results were presented. The ratio of the absorbance of cells treated with samples obtained from Sisymbrium spp. and isopropyl isothiocyanate and the absorbance of those that are not treated is an indicator of the cytotoxic activity of the samples and isopropyl isothiocynate we used. The most significant cytotoxic effect on the A549 cell line was achieved after 72 h of incubation, but there was no significant difference in the action of the samples obtained by different isolation methods. The most significant cytotoxic effect is achieved after treatment of MDA-MB-231 cell line with Sisymbrium orientale sample obtained by silique autolysis at a concentration of 100 µg/mL after 72 hours of incubation. Number of metabolically active cells decreased to 38%. Sisymbrium orientale sample obtained by silique autolysis after 72 h of incubation at a concentration of 100 μg/mL showed the most significant cytotoxic effect on the T24 cell line. as well, but slightly weaker. Samples obtained from Sisymbrium spp. and isopropyl isothiocynate showed a cytotoxic effect dependent on the incubation time and concentration. The effectiveness of samples obtained from Sisymbrium spp. and isopropyl isothiocynate was not always correspondent to the increase of concentration and incubation time and, in some cases, cell recovery occurred. Isopropyl isothiocyanate has a weak cytotoxic effect on breast cancer cell lines (MDA-MB-231) and bladder cancer (T24) only at a maximum concentration of 100 µg/mL after 72 h of incubation.