<p>While arbuscular mycorrhizal fungi (AMF) influence plant nitrogen (N) acquisition and drought tolerance, the complex interactions governing N uptake under varying water deficits remain unclear. To addressing this, well-characterized mycorrhizal tomato type (MYC) and its mycorrhiza-defective mutant (referred to as rmc) were labeled with ¹⁵NH₄Cl under normal and drought conditions in a greenhouse. We quantified ¹⁵N allocation in plant biomass and rhizosphere soil, while assessing shifts in activities of β-N-acetylglucosaminidase and leucine aminopeptidase. MYC increased tomato total biomass relative to rmc under both water regimes, with the most pronounced root biomass enhancement observed under drought (64–74%). Similarly, MYC increased tomato ¹⁵N uptake compared to rmc, with a greater increase under drought (80–104%) versus normal (55–94%) conditions. This phenomenon can be ascribed to elevated ¹⁵N enrichment in microbial biomass and increased activities of β-N-acetylglucosaminidase and leucine aminopeptidase. This was further supported by the positive correlations between tomato ¹⁵N acquisition and ¹⁵N incorporation into microbial biomass as well as activities of β-N-acetylglucosaminidase and leucine aminopeptidase. Collectively, AMF alleviated drought stress and improved plant productivity through enhanced root N-acquisition capacity, increased microbial biomass and enzyme secretion, and optimized soil N mineralization processes.</p>