The rhizosphere and the hyphosphere are critical interfaces for plant–microbe interactions. It is essential to understand how extracellular enzyme activities and microbial biomass vary across these compartments in order to predict soil biogeochemical cycling under different nutrient regimes. To investigate this, we conducted a pot experiment with wheat grown in soils from a century-old fertilization trial. A 41-μm nylon mesh was used to physically separate the rhizosphere from the hyphosphere, enabling the measurement of extracellular enzyme activities, microbial biomass and available nutrient concentrations in the two compartments. We found that while rhizosphere enzyme activities generally increased by up to 90 %, the hyphosphere enzyme activities decreased by up to 35 % compared to the initial soil. These differences were not accompanied by corresponding changes in microbial biomass carbon. This pronounced spatial variation depended heavily on the fertilization regime. The greatest difference occurred with mineral NPK fertilization, where enzyme activities were 43–91 % higher in the rhizosphere than in the hyphosphere. In contrast, the use of combined mineral and organic fertilization eliminated this spatial difference, with similar activities observed in both compartments. Crucially, these variations were more strongly associated with initial phosphorus availability than with carbon availability. These findings suggest that phosphorus availability plays a central role in shaping the spatial distribution of enzyme activity across soil compartments.