The identification of enzyme inhibitors from natural sources offers a promising pathway for drug discovery. In this study, affinity selection-mass spectrometry (AS-MS) was employed to screen for inhibitors of Leishmania donovani nucleoside hydrolase (NH) from crude extracts of Banisteriopsis laevifolia. The enzyme was immobilized onto magnetic nanoparticles, enabling selective ligand retention and downstream analysis. The leaf extract exhibited significant inhibitory activity, with an IC value of 0.73 ± 0.09 μg/mL, prompting further exploration. Analytical-scale fractionation and biochromatogram analysis revealed inhibitory regions, while AS-MS facilitated the annotation of nine flavonoid-based ligands, including procyanidins, glycosylated flavonoids, and rutin. The structures of four ligands (isoquercetin, astragalin, rutin, and orientin) were confirmed using commercial standards. Among these, isoquercetin and astragalin demonstrated potent NH inhibition with IC values of 40.2 ± 16.6 and 41.6 ± 8.9 μmol/L, respectively. These findings highlight B. laevifolia as a promising source of bioactive compounds and demonstrate the utility of AS-MS for efficiently identifying enzyme inhibitors in natural libraries.