The fishing cat (Prionailurus viverrinus) is a medium-sized wetland specialist threatened felid with a limited knowledge base on behavior and ecology. We carried out a comprehensive analysis of its diet within a human-dominated landscape in southern Nepal, analyzing scat samples through microscopic analysis and DNA metabarcoding. The microscopic analysis revealed that mammals, primarily rodents, constituted the largest proportion of fishing cat diet (61.73% frequency of occurrence (FO)) followed by birds (43.21% FO) fish (24.69% FO), and reptiles (19.75% FO). However, DNA metabarcoding documented fish as the main diet (70% FO), followed by mammals (63.33% FO) and birds (30% FO). Our findings demonstrate that fish alone is not a major food source of fishing cats indicating their role in regulating rodents and birds that damage farm crops. The trophic niche breadth (0.68) shows the fishing cats a diet generalist. We suggest that highlighting these findings through community education and awareness can change farmers’ negative perceptions, reducing threats of retaliatory killing and increasing support for conservation efforts.