<p>Little is known about the path of root-derived carbon (C) into soil microbial communities in response to arbuscular mycorrhizal fungi (AMF) and nitrogen (N) fertilization. A mycorrhiza defective mutant of tomato (reduced mycorrhizal colonization: <i>rmc</i>) and its mycorrhizal wild type progenitor (MYC) were used to control for the formation of AMF. 16-week continuous ¹³CO₂ labeling was performed to quantify the photosynthetic C allocation in active microorganisms via ¹³C profiles of neutral (NLFAs) and phospholipid fatty acids (PLFAs). The ¹³C incorporation into fungal biomarker (the sum of PLFA 16:1ω5c, NLFA 16:1ω5c, PLFA 18:2ω6,9) increased with time over 16 weeks, and 4.62% of totally assimilated C was incorporated into AMF. More ¹³C was allocated into AMF storage compounds (NLFA 16:1ω5c, 3.1–4.1%) than hyphal biomass (PLFA 16:1ω5c, 0.12–0.25%). Furthermore, AMF symbiosis shifted microbial community composition, resulting in a lower ¹³C incorporation into bacteria and saprotrophic fungi compared to <i>rmc</i> plants. This suggests a lower use of root-derived C by bacteria and saprotrophic fungi but preference to older C compounds as energy sources. However, N fertilization decreased AMF abundance and subsequently less root-derived C was incorporated into PLFA and NLFA 16:1ω5c in relative to unfertilized soils, due to less C allocation caused by an increased C immobilization in the aboveground biomass. Our findings suggested that root-derived C can be sequestered by AMF through storage in their reproductive organs, but the preferential C allocation to AMF might be at the expense of C flow to other microbial groups. Overall, our results confirmed that mycorrhizal plants exert a greater influence on C incorporation into bacteria and saprotrophic fungi, which, however, is highly dependent on N fertilization.</p>