Rain gardens are an alternative to traditional drainage, able to lower flood risk and reduce environmental contamination from stormwater. Removal of contaminants by rain gardens is driven by both physical processes (such as filtration and sedimentation) and biological metabolic processes by soil microorganisms. To better understand rain garden performance, this study explored the impact of rain gardens on pollution removal and microbial composition and function using rain gardens fed real stormwater from a busy road. Each rain garden had different grain size and hydraulic conductivities as these parameters have been argued to impact pollution removal. All four rain gardens were able to reduce the contaminant load in the stormwaters, reducing the concentration of dissolved metals, suspended solids and chemical oxygen demand. Significantly, road salting in the winter did not cause dissolved metals to be released from the rain gardens, suggesting that rain gardens can continue to reduce contaminant loads during winter salting regimes. Some variation in pollutant removal was seen between the soils tested, but overall no clear trend could be identified based on grain size and hydraulic conductivity with all rain gardens performing broadly similarly. The rain garden soil altered the microbial community in the stormwater, resulting in greater taxonomic evenness and functional richness in the effluent water compared to the influent. Functional richness of the soils was also higher than that of the input waters, indicating that the microbes in the rain gardens were able to perform a wider range of functions than those of the influent. Effluent and soil microbiology was more impacted by sampling date than soil grain size, which may be a result of the soil communities maturing and changing over time. As greater numbers of rain gardens are installed to tackle flooding from climate change, it is important to ensure the environment is protected from urban contaminants in the stormwater. The results in this study further highlight the ability of rain gardens to undertake this important task.