This study presents new inhibitors of the nucleoside hydrolase from Leishmania donovani (LdNH) with in vitro leishmanicidal activity. Biological screening of 214 Brazilian plant extracts was performed to select plants with enzyme inhibitory activity. Two plants were selected for their results, and for their lack of prior phytochemical description: Leandra amplexicaulis DC. (Melastomataceae) and Urvillea rufescens Cambess (Sapindaceae). Three flavonoids were isolated by bioguided fractionation of the hydroethanolic extracts: kaempferol 3-O-α-L-rhamnopyranoside (1) and kaempferol 3-O-β-D-xylopyranosyl-(1→2)-α-L-rhamnopyranoside (2) from L. amplexicaulis, as well as tricetin-4′-O-methyl flavone (3) from U. rufescens. These flavonoids showed inhibitory activities (IC₅₀) of 197.4 μM (1), 74.7 μM (2) and 1.1 μM (3) on the LdNH. Their binding mode was proposed based on molecular docking with LdNH and by NMR Saturation Transfer Difference studies. Kinetic studies demonstrate that the most potent inhibitor (3) acts by uncompetitive inhibition. This study reports for the first time the inhibition of LdNH by naturally sourced flavonoids.