Understanding the diets of carnivores is essential for resolving food web interactions and the population dynamics of both prey and predators and for designing effective strategies for species and ecosystem conservation. As effective predators, wild felids play important roles in various ecosystems, but relatively little is known about the dietary habits of many species, primarily owing to their elusive behavior. We used a DNA-based method to analyze the vertebrates and plants constituting the diet of leopard cats (Prionailurus bengalensis) in the temperate forests of the mountains of southwest China, a global biodiversity hotspot. DNA extracted from leopard cat scats was amplified with primers targeting either the vertebrate mitochondrial 12S rRNA gene (N = 25 scats) or the psbCL region of the chloroplast genome of vascular plants (N = 42 scats). The polymerase chain reaction products were sequenced and prey taxa were assigned based on sequence similarity. We identified a total of 16 taxa of vertebrate prey, with pikas (in 76% of the scats) and rodents (40%) predominating. Plant material belonging to 12 taxa was found in 76% of the samples, and the genus Solanum and subfamily Rosoideae were the most frequently detected plant taxa. The frequency of occurrence of identified plant taxa differed between the spring–summer and fall–winter months. Thus, the leopard cats in our study area have a diversified diet and are highly flexible in adapting their foraging behavior to the locally available prey. Our data suggest that preserving their natural prey base dominated by pikas and rodents may be vital for the subsistence of local populations. The high species resolution and detection sensitivity of the DNA-based method we used in this study make it a powerful and efficient tool for fine-scale analysis of complex diets.