The isothiocyanates present in the cruciferous plants were proved to have the antiproliferative and cytotoxic effect on cancer cell lines. Natural compounds in combination with chemotherapy agents enhance anticancer activities of drugs and reduce their toxicity. The aim of the presented study was to determine an effect of isothiocyanates and 5-fluorouracil used alone or in combination (in sequential or co-administrative treatments) on normal cell lines-V79. There were compared abilities of three isothiocyanates to interact with 5-fluorouracil. There was also investigated the mechanism of interaction and influence of isothiocyanates on 5-fluorouracil. The cell survival was evaluated with MTT assay. Combination effects between isothiocyanates and 5-fluorouracil were estimated in the way described by Chou and Talalay. The cycle progression and the living cells number were determined with flow cytometry. The type of cell death was detected with a confocal microscope. There was observed an antagonistic effect which was mainly dependent on the cell cycle distribution e.g., sulforaphane increased the cell number in the G2/M phase, whereas 5-fluorouracil and combination of these two compounds increased the cell number in the S phase. If each compound blocked the S phase of the cell cycle, their combination increased the cell number in the S phase, but the increase was not statistically significant when compared with single substance treatments. The highest antagonistic effect in normal cells was obtained for co-administrated 5-fluorouracil and 2-oxoheptyl isothiocyanate at the fraction affected at 0.5 and 0.75. Isothiocyanates did not affect 5-fluorouracil cytotoxicity in normal cell lines-V79.