Soil organic matter (SOM) content is a key indicator of riparian soil functioning and in the provision of ecosystem services such as water retention, flood alleviation, pollutant attenuation and carbon (C) sequestration for climate change mitigation. Here, we studied the importance of microbial biomass and nutrient availability in regulating SOM turnover rates. C stabilisation in soil is expected to vary both vertically, down the soil profile and laterally across the riparian zone. In this study, we evaluated the influence of five factors on C mineralization (Cmin): (i) substrate quantity, (ii) substrate quality, (iii) nutrient (C, N and P) stoichiometry, (iv) soil microbial activity with proximity to the river (2 to 75 m), and (v) as a function of soil depth (0 – 3 m). Substrate quality, quantity and nutrient stoichiometry were evaluated using high and low molecular weight 14C-labelled dissolved organic (DOC) along with different nutrient additions. Differences in soil microbial activity with proximity to the river and soil depth were assessed by comparing initial (immediate) Cmin rates and cumulative C mineralized at the end of the incubation period. Overall, microbial biomass C (MBC), organic matter (OM) and soil moisture content (MC) proved to be the major factors controlling rates of Cmin at depth. Differences in the immediate and medium-term response (42 days) of Cmin suggested that microbial growth increased and carbon use efficiency (CUE) decreased down the soil profile. Inorganic N and/or P availability had little or no effect on Cmin suggesting that microbial community growth and activity is predominantly C limited. Similarly, proximity to the watercourse also had relatively little effect on Cmin. This work challenges current theories suggesting that areas adjacent to watercourse process C differently from upslope areas. In contrast, our results suggest that substrate quality and microbial biomass are more important in regulating C processing rates rather than proximity to a river.