Non-antibiotic conditions, including organophosphorus pesticides (OPPs), have been implicated in the horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) to varying degrees. While most studies focus on the toxicity of OPPs to humans and animals, their roles in ARG dissemination remain largely unexplored. In this study, we investigate the effects and involved molecular mechanisms of environmentally relevant concentrations of malathion and dimethoate, two representative OPPs, on plasmid-mediated conjugal transfer. By detecting reactive oxygen species (ROS) production and cell membrane permeability, we gained insights into the underlying processes. Furthermore, we substantiated the role of ROS and cell membrane permeability in plasmid-mediated conjugative transfer through the analysis of relevant antioxidant enzyme activities, cell membrane-related indices, and RNA sequences. Additionally, our examination of proton motive force and adenosine triphosphate content provided evidence that OPPs create conditions conducive to plasmid-mediated conjugative transfer from an energetic perspective. The findings of the present study highlight the potential risk of OPPs in promoting ARG spread, which could ultimately provide new theoretical support and direction for future research on the impacts of pesticides on ARG propagation.