Despite recognising rain gardens as a best management practice (BMP) to mitigate urban stormwater runoff, there is a dearth of knowledge about their treatment and infiltration performance. It is believed that organic substrates may enhance some contaminant removal but hinder hydraulic throughput although data showing this is sparse. In order to evaluate the influence of substrate composition on bioinfiltrative system effectiveness, mesocosm-scale (180 L, 0.17 m2) laboratory rain gardens were established. Saturated (constant head) hydraulic conductivity was determined before and after the experimental treatment tests that employed stormwater collected from a neighbouring catchment to investigate contaminant removal efficiencies. The principal contaminant (Zn, Cu, Pb and nutrients) removal efficiencies were investigated for three substrates comprising various proportions of organic topsoil. All total metal concentrations in the effluent were <50% of influent concentrations, with the exception of copper in the topsoil-only system that had negligible reduction due to a high dissolved fraction. The system comprising topsoil only had the lowest saturated hydraulic conductivity of 162 mm/hr and demonstrated the poorest metal (Cu, Zn) removal efficiencies. Interestingly, the system with a combination of sand and topsoil demonstrated most promising metal removal of Cu (53%), Zn (81.2%) and Pb (89.1%) with adequate hydraulic performance (296 mm/hr) required for a stormwater infiltrative system. Overall, metal removal was greater at an effluent pH of 7.38 compared to the 6.24 pH provided in the raw stormwater. Some pH buffering was provided by the calcareous sand in two of the systems, whereas the topsoil-only system lacked such buffering potential to facilitate adequate metal removal. These data highlight the influence of organic topsoil on pH that clearly governs metal speciation and hence removal efficacy in bioinfiltrative systems. Nitrate was net exported from all the systems, especially topsoil contrary to what is believed to be easily removed.