Dichlorodiphenyltrichloroethane (DDT) is an organochloride pesticide with a global impact on agriculture. Despite the international ban on this pesticide in 2001, DDT is still illegally used for malaria control in some parts of the world, such as India and South Africa. The presence of DDT and its metabolites in waters, soil, and sediments continue to adversely affect living organisms. Understanding the toxic effects of DDT is crucial for global environmental health. The goal of this study was to investigate the toxicity of DDT in E. coli ATCC-25922, Sarcina spp., ATCC-35659, Enterobacter homaechei LBM ATCC-700323, Staphylococcus aureus ATCC-25923 and Candida albicans ATCC-1023. To confirm oxidative stress as a mechanism of toxicity, sodA promoter induction in the Escherichia coli sodA:luxCDABE biosensor strain and ROS (Radical Oxygen Species) synthesis in E. coli ATCC-25922 strain, across various DDT concentrations (10, 1, 0.1, and 0.01 mg/L) were measured. The results showed that DDT in the range of applied concentrations shows a toxic effect on bacteria/fungi. Analysis of sodA promoter induction and ROS synthesis values in E. coli strains showed an increase in these indicators following exposure to the tested DDT concentrations. The results confirmed the validity of the hypothesis that the molecular mechanism of DDT toxicity is by induction of oxidative stress. Therefore, the need to develop more effective methods of removing DDT from wastewater and water and reducing the transport of this pesticide into the environment is justified. Moreover, recent evidence has increasingly confirmed positive correlations between human exposure to DDT and the development of cancers.