For a study, it was determined that antibiotic-resistant bacteria (ARB) were a significant reservoir in the gut microbiota, posing a serious hazard to public health. However, little was known regarding ARB’s development in the gut following a combination of antibiotic and non-antibiotic pharmaceutics exposure. The antidepressant duloxetine (2.5 µg/L–25 mg/L) and chloramphenicol (6 µg/L–4 mg/L) were given to Escherichia coli, a frequent opportunistic pathogen in the gut microbiota. To assess the minimal inhibitor concentration (MIC) of 29 antibiotics, resistant bacteria were obtained. The synergy between duloxetine and chloramphenicol was then quantified using genome-wide DNA sequencing, global transcriptome sequencing, and real-time quantitative polymerase chain reaction. Compared with independent exposure, combined exposure raised the mutation frequency of chloramphenicol resistance by 2.45–9.01 fold. The presence of substantial duloxetine and chloramphenicol synergy was suggested by a combination index of 187.7. The mutants that resulted had heritable increased resistance to 12 antibiotics and were ARB to 8 others. In addition, combined exposure raised the transcriptome expression of acrA, acrB, and marA in E. coli, resulting in a more robust oxidative stress response. Greater triggers to the AcrAB-TolC transport system and the MlaFEDB ABC transporter via reactive oxygen species (ROS)-induced mutagenesis, verified by gene knockout, contributed to the synergistic enhancement of antibiotic resistance in the combined exposure group, along with the occurrence of DNA mutations in Marr in the mutants. The E. coli mutants revealed 1.1–1.7-fold increases in the expression levels of acrA, acrB, acrZ, mdtE, and mdtF, regardless of whether their formation was driven by duloxetine, chloramphenicol, or a combination of the 2. The pattern suggested that the mutants had the exact chloramphenicol resistance mechanisms, which involved the enhanced efflux pumps AcrAB-TolC and mdtEF. The data showed that antibiotics and non-antibiotic pharmaceutics worked together to speed up the evolution of ARB and might have increased their dissemination.