Bioretention basins are one of the most commonly used green stormwater features, with the potential to accumulate significant levels of nitrogen (N) in their soil and to permanently remove it through denitrification. Many studies have investigated the N removal potential of bioretention basins through the assessment of inflow and outflow concentrations. However, their long-term N removal through soil accumulation and denitrification potential is less known. This study investigated the temporal variation of total N (TN) accumulation and denitrification potential in soils of 25 bioretention basins within a 13-year soil chronosequence, in a subtropical climate in Australia. The denitrification potential of a subset of seven bioretention basins was investigated in accompaniment with nutrient and soil characteristics. Additionally, stable isotopes (δ13C and δ15N) were used to assess temporal changes in the soil composition as well as to identify the sources of carbon (C) into these basins. Over 13 years of operation, TN accumulated faster in the top 5 cm of soil than deeper soils. Soil TN density was highest in the top 5 cm with an average of 1.4 kg N m−3, which was about two times higher than deeper soils. Site age and soil texture were the best predictors of soil TN density and denitrification (1 to 9.7 mg N m−2 h−1). The isotope values were variable among basins. Low δ15N values in young basins (-1.02 ‰) suggested fixation as the main source of N, while older basins had higher δ15N, indicating higher denitrification. Bioretention plants were the primary source of soil C; although the occurrence of soil amendment also contributed to the C pool. To improve the performance of these bioretention basins, we recommend increasing vegetation at initial years after construction, and enhancing more frequent anaerobic conditions in the high soil profile. These two conditions can improve denitrification potential, and thus the performance of these basins for improving water quality.