WWTPs have been regarded as an important hot spot of ARGs. The discharge point of WWTP effluent, where ARGs may be horizontally transferred from bacteria of treated wastewater to bacteria of receiving water, is an important interface between the human-dominated ecosystem and the natural environment. The use of batch cultures in previous studies cannot adequately simulate the nutrient conditions and growth rates in receiving water. In this study, chemostats were employed to simulate the continuous growth of bacteria in receiving water. Furthermore, the experimental setup allowed for separate investigations on the effects of nutrient levels (i.e., simulating background nutrients in river water) and bacterial growth rates on conjugation frequencies and resulting resistance levels. The study generates statistically sound ecological data that can be used to estimate the risk of wastewater-originated ARGs as part of the One Health framework.