Impacts of climate change drivers on soil biota and biogeochemical cycling are of growing concern. Soil nitrogen (N) availability has the potential to mediate microbial responses to changes in precipitation via changes in microbial community composition, but the interactions between N spatial pattern and rainfall regime on soil microbial community structure and function remain unclear. We conducted an outdoor mesocosm experiment with a model grassland community to assess interactive effects of rainfall amount (control, −50% rainfall, +50% rainfall) and N spatial pattern (homogeneous, heterogeneous) on bacterial carbon (C) use and functional diversity using a community-level physiological profiling approach. Patchy N addition increased total bacterial C use and diversity of substrate use irrespective of rainfall treatment. Observed increases in microbial functional diversity contrasted with patch-induced decreases in plant community diversity in the study system. Neither bacterial C use nor diversity of C sources showed a significant response to rainfall regime or a significant rainfall × N pattern interaction. However, Principal Components Analysis showed clustering of bacterial substrate use across treatments, in line with different strategies of C use linked to both N spatial pattern and rainfall regime. Our findings suggest that bacterial substrate profiles are more sensitive to dry conditions when soil N is patchily distributed, with significant implications for soil organic C composition and storage potential in heterogeneous grasslands under future climatic conditions.