The use of organophosphate pesticides, such as profenofos, is a pressing environmental concern due to their persistence and toxicity to non-target organisms. For this reason, developing alternatives to remove them from the environment is crucial. The objective of this study was to biosynthesize ZnO nanoparticles (ZnO NPs) using an aqueous extract of Negra Criolla grape seeds for their application in the degradation of profenofos in synthetic solutions. The biosynthesized ZnO NPs had an average size of 52 ± 2 nm and a maximum absorption at 375 nm, characteristic of the surface plasmon resonance of ZnO NPs. The ATR-FTIR spectra showed peaks characteristic of ZnO NPs. The 2² × 3 factorial design showed that the optimal values for pH and ZnO NPs concentration are 5 and 3 g/L, respectively, achieving a primary degradation of profenofos (20 mg/L) of 59 ± 2% after 120 min of UV irradiation. In conclusion, it was demonstrated that ZnO NPs biosynthesized using an aqueous extract of Negra Criolla grape seeds exhibit photocatalytic activity for the degradation of profenofos; however, further studies are needed to evaluate their application in actual contaminated water.