The current study was emphasized to assess the effect of malathion on root system (cell division and kinetics of the root elongation) and stress related parameters in Allium cepa L. The roots were exposed to different concentrations (0.05, 0.13, 0.26, 0.39 and 0.52 g/L) of malathion for different treatment periods (4, 8 and 18 h). The results revealed that malathion application affected the growth rate and cell division in root tips. The root elongation kinetics were impaired at 0.13 to 0.52 g/L concentrations. Reduction in tissue water content (TWC) indicated the limited osmotic adjustment due to membrane damage. Further, a decrease in sucrose content was observed in contrast to the accumulation of proline (upto 0.39 g/L). Moreover, malathion exposure elevated the levels of lipid peroxidation followed by changes in antioxidant enzymes status. The activities of ascorbate peroxidase (APX) and glutathione reductase (GR) were down-regulated whereas the activities of catalase (CAT), glutathione-S-transferase (GST) and superoxide dismutase (SOD) were up-regulated except in 0.52 g/L malathion. The molecular docking study of malathion with CAT, GST, SOD, APX and GR also supported of above results for their activity. All these physiological responses varied with increasing malathion concentration and duration of treatment. The single cell gel electrophoresis results showed that all concentrations of malathion induced DNA damage in root cells. The findings depicted that malathion application induces cytotoxic and phytotoxic effects mediated through oxidative stress and subsequent injuries.