•Diuron metabolites were toxic in human cancer cell lines BeWo, MCF-7 and Caco-2.•Clear toxicity by MTT assay indicates mitochondrial-mediated toxicity.•In comparison between the cell lines, MCF-7 was the most resistant.•Increased ROS may not explain cytotoxicity of diuron metabolites. Diuron, a highly used herbicide worldwide, is metabolized into several toxic metabolites. DCA (3,4-dichloroaniline), DCPU 3-(3, 4-dichlorophenyl)urea and DCPMU 3-(3,4-dichlorophenyl)-1-methyl urea reduced viability of human placental choriocarcinoma BeWo, human breast adenocarcinoma MCF-7 and human colon adenocarcinoma Caco-2 cells as judged by the MTT assay, where color formation is dependent on functional mitochondria in viable cells. Based on the IC50 values in BeWo cells the order of cytotoxicity was DCA > DCPU > diuron > DCPMU, and in Caco-2 cells DCPMU > DCPU > DCA, diuron. In MCF-7 cells, only DCPU had an IC50 within the range of the concentrations used. In the PI-digitonin viability assay, only the highest concentration (200 μM) of DCPU caused a statistically significant decrease in viability in any cell line. There was no correlation between cytotoxicity and ROS production. This indicates that diuron metabolites are toxic in cells of human origin with mitochondria as the target, but ROS not the likely mechanism.