In a continuation of our computational efforts to find new natural inhibitors of a variety of target enzymes from parasites causing neglected tropical diseases (NTDs), we now report on 15 natural products (NPs) that we have identified as inhibitors of pteridine reductase I (PTR1) through a combination of in silico and in vitro investigations. Pteridine reductase (PTR1) is an enzyme of the trypanosomatid parasites' peculiar folate metabolism, and has previously been validated as a drug target. Initially, pharmacophore queries were created based on four 3D structures of PTR1 using co-crystallized known inhibitors as templates. Each of the pharmacophore queries was used to virtually screen a database of 1100 commercially available natural products. The resulting hits were submitted to molecular docking analyses in the substrate binding site of the respective protein structures used for the pharmacophore design. This approach led to the in silico identification of a total of 18 NPs with predicted binding affinity to PTR1. These compounds were subsequently tested in vitro for inhibitory activity towards recombinant PTR1 in a spectrophotometric inhibition assay. Fifteen out of the 18 tested compounds (hit rate = 83%) showed significant inhibitory activity against PTR1 when tested at a concentration of 50 µM. The IC values were determined for the six NPs that inhibited the target enzyme by more than 50% at 50 µM, with sophoraflavanone G being the most active compound tested (IC = 19.2 µM). The NPs identified and evaluated in the present study may represent promising lead structures for the further rational drug design of more potent inhibitors against PTR1.