Abstract: Residual disinfectants in aquatic environments may influence the dissemination of antibiotic resistance genes (ARGs) by affecting horizontal gene transfer among microorganisms. The phenolic disinfectant p-chloro-m-xylenol (PCMX) has been widely used and is frequently detected in municipal wastewater and surface waters at concentrations ranging from ng/L to μg/L. However, its potential effects on plasmid-mediated ARG dissemination remain insufficiently understood. This study investigated the influence of PCMX on the conjugative transfer of ARGs and explored the underlying mechanisms involved. An intergeneric conjugation model was established using Escherichia coli CHS56 carrying plasmid RP4-8 as the donor strain and Pseudomonas sp. TS44 as the recipient strain. The effects of PCMX on conjugative transfer were evaluated across a concentration range of 0.1–10,000.0 μg/L. Conjugation experiments were combined with reactive oxygen species (ROS) detection, cell membrane permeability measurements, and transcriptomic analysis to investigate the physiological responses and molecular mechanisms associated with PCMX exposure. The results demonstrated that PCMX promoted plasmid-mediated conjugative transfer within an environmentally relevant concentration range. Specifically, at concentrations between 0.1 and 100.0 μg/L, PCMX enhanced the transfer frequency of plasmid RP4-8 between the donor and recipient strains, reaching a peak conjugation frequency of 7.22 × 10⁻⁶ at 10 μg/L PCMX. When the concentration increased to 1,000–10,000 μg/L, the conjugation frequency declined, which may be attributed to the inhibitory effects of high PCMX concentrations on bacterial growth and cellular activity. Flow cytometry analysis using DCF-DA staining indicated that PCMX exposure increased intracellular ROS levels in the donor strain E. coli CHS56 at 100 and 10,000 μg/L. In contrast, no significant ROS variation was detected in the recipient strain Pseudomonas sp. TS44. The addition of the ROS scavenger N-acetylcysteine (NAC) significantly reduced intracellular ROS levels in the donor strain but did not markedly decrease the conjugation frequency, suggesting that oxidative stress was not the dominant driver for the enhancement of plasmid transfer under PCMX exposure. Membrane permeability analysis revealed that PCMX exposure altered the integrity and permeability of bacterial cell membranes, particularly in the recipient strain. Increased membrane permeability likely facilitated physical contact between donor and recipient cells, thereby promoting plasmid transfer. Transcriptomic analysis further showed that PCMX exposure significantly affected the global gene expression profiles of both strains. Multiple genes associated with membrane transport and transmembrane processes were differentially expressed. Notably, a glycerol uptake facilitator gene involved in membrane transport was significantly upregulated, indicating enhanced transmembrane transport activity under PCMX stress. Overall, these results suggest that PCMX enhancea plasmid-mediated ARG conjugative transfer primarily by altering cell membrane permeability and transmembrane transport processes rather than through ROS-mediated oxidative stress. Considering that PCMX cannot be completely removed during conventional wastewater treatment, residual PCMX may persist in biological treatment units containing dense microbial communities, thereby increasing the potential risk of ARG dissemination. These findings provide experimental evidence for evaluating the ecological risks of disinfectant residues in wastewater treatment systems and aquatic environments.