The emergence of clinically relevant β-lactam-resistant bacteria poses a serious threat to human health and presents a major challenge for medical treatment. How opportunistic pathogenic bacteria acquire antibiotic resistance and the prevalence of antibiotic-resistant opportunistic pathogenic bacteria in the environment are still unclear. In this study, we further confirmed that the selective pressure of heavy metals contributes to the increase in ampicillin-resistant opportunistic pathogens in the Xiangjiang River. Four ampicillin-resistant opportunistic pathogenic bacteria (Pseudomonas monteilii, Aeromonas hydrophila, Acinetobacter baumannii, and Staphylococcus epidermidis) were isolated on Luria-Bertani (LB) agar plates and identified by 16S rRNA sequencing. The abundance of these opportunistic pathogenic bacteria significantly increased in the sites downstream of the Xiangjiang River that were heavily influenced by metal mining activities. A microcosm experiment showed that the abundance of β-lactam resistance genes carried by opportunistic pathogenic bacteria in the heavy metal (Cu2+ and Zn2+) treatment group was 2–10 times higher than that in the control. Moreover, heavy metals (Cu2+ and Zn2+) significantly increased the horizontal transfer of plasmids in pathogenic bacteria. Of particular interest is that heavy metals facilitated the horizontal transfer of conjugative plasmids, which may lead to the prevalence of multidrug-resistant pathogenic bacteria in the Xiangjiang River. Display omitted •Ampicillin-resistant pathogenic bacteria were isolated and identified in the Xiangjiang River.•The pathogenic bacteria cover P. monteilii, A. hydrophila, A. baumannii, and S. epidermidis.•The selective pressure of heavy metals contributed to the bloom of the pathogenic bacteria.•Cu2+ and Zn2+ increased the horizontal transfer of plasmids in the pathogenic bacteria.•Heavy metals may ultimately cause the prevalence of multidrug-resistant pathogenic bacteria. This study provided a reference for the effects of selective stress of heavy metal pollutants on the prevalence of ampicillin-resistant opportunistic pathogenic bacteria.