Root exudation increases the concentration of readily available carbon (C) compounds in its immediate environment. This creates 'hotspots' of microbial activity characterized by accelerated soil organic matter turnover with direct implications for nutrient availability for plants. However, our knowledge of the microbial metabolic processes occurring in the immediate vicinity of roots during and after a root exudation event is still limited. Using reverse microdialysis, we simulated root exudation by releasing a 13 C-labelled mix of low-molecularweight organic C compounds at mm-sized locations in undisturbed soil. Combined with stable isotope tracing, we investigated the fine-scale temporal and spatial response of microbial metabolism, soil chemistry, and traced microbial respiration and uptake of exuded compounds. Our results show that a 9-h simulated root exudation pulse leads to i) a large local respiration event and ii) alteration of the temporal dynamics of soil metabolites over the following 12 day at the exudation spot. Notably, we observed a threefold increase in ammonium concentrations at 12 h and increased nitrate concentrations five days after the pulse. Moreover, various short-chain fatty acids (acetate, propionate, formate) increased over the following days, indicating altered microbial metabolic pathways and activity. Phospholipid and neutral lipid fatty acids (PLFAs, NLFAs) of all major microbial groups were significantly 13 C-enriched within a 5 mm radius around the microdialysis probes, but not beyond. The highest relative 13 C enrichment was observed in fungal NLFAs, indicating that a significant proportion of the exuded compounds had been incorporated into fungal storage compounds. Our findings indicate that the punctual release of low-molecular-weight organic C compounds into intact soil significantly changes microbial metabolism and activity in its immediate surroundings, enhancing mineralization of native organic nitrogen. This highlights the versatility of microbial metabolic pathways in response to rapidly changing C availability and their effectiveness in increasing nutrient availability near plant roots.