Nitrate (NO₃^-) leaching is a serious health and ecological concern in global agroecosystems, particularly those under the application of agricultural-managed aquifer recharge (Ag-MAR); however, there is an absence of information on microbial controls affecting NO₃^- leaching outcomes. We combine natural dual isotopes of NO₃^- (¹⁵N/¹⁴N and ¹⁸O/¹⁶O) with metagenomics, quantitative polymerase chain reaction (PCR), and a threshold indicator taxa analysis (TITAN) to investigate the activities, taxon profiles, and environmental controls of soil microbiome associated with NO₃^- leaching at different depths from Californian vineyards under Ag-MAR application. The isotopic signatures demonstrated a significant priming effect (P < 0.01) of Ag-MAR on denitrification activities in the topsoil (0-10 cm), with a 12-25-fold increase of ¹⁵N-NO₃^- and ¹⁸O-NO₃^- after the first 24 h of flooding, followed by a sharp decrease in the enrichment of both isotopes with ∼80% decline in denitrification activities thereafter. In contrast, deeper soils (60-100 cm) showed minimal or no denitrification activities over the course of Ag-MAR application, thus resulting in 10-20-fold of residual NO₃^- being leached. Metagenomic profiling and laboratory microcosm demonstrated that both nitrifying and denitrifying groups, responsible for controlling NO₃^- leaching, decreased in abundance and potential activity rates with soil depth. TITAN suggested that Nitrosocosmicus and Bradyrhizobium, as the major nitrifier and denitrifier, had the highest and lowest tipping points with regard to the NO₃^- changes (P < 0.05), respectively. Overall, our study provides new insight into specific depth limitations of microbial controls on soil NO₃^- leaching in agroecosystems.