Pteridine reductase 1 (PTR1) is a trypanosomatid multifunctional enzyme that provides a mechanism for escape of dihydrofolate reductase (DHFR) inhibition. This is because PTR1 can reduce pterins and folates. Trypanosomes require folates and pterins for survival and are unable to synthesize them de novo. Currently there are no anti-folate based Human African Trypanosomiasis (HAT) chemotherapeutics in use. Thus, successful dual inhibition of dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1) has implications in the exploitation of anti-folates. We carried out molecular docking of a ligand library of 5742 compounds against PTR1 and identified 18 compounds showing promising binding modes. The protein-ligand complexes were subjected to molecular dynamics to characterize their molecular interactions and energetics, followed by in vitro testing. In this study, we identified five compounds which showed low micromolar Trypanosome growth inhibition in in vitro experiments that might be acting by inhibition of PTR1. Compounds RUBi004, RUBi007, RUBi014, and RUBi018 displayed moderate to strong antagonism (mutual reduction in potency) when used in combination with the known DHFR inhibitor, WR99210. This gave an indication that the compounds might inhibit both PTR1 and DHFR. RUBi016 showed an additive effect in the isobologram assay. Overall, our results provide a basis for scaffold optimization for further studies in the development of HAT anti-folates.