Objective To clarify the pathogen causing Trichosanthes kirilowii fruit rot and screen effective fungicides for controlling the disease. Method Samples of rot fruit from T. kirilowii plants were collected from Dabie Mountain Areas, Anhui Province in 2019—2020. A total of 35 representative isolates were obtained by tissue isolation. The pathogenicity was verified according to the Koch’s postulate, and the causal agents were identified based on the morphological characteristics and multilocus sequence analysis. The indoor toxicity of seven fungicides against the pathogen was detected by the mycelium growth rate method. Result Colonies with four different morphological characteristics were isolated and purified. The isolates were confirmed to be pathogenic to the fruit of T. kirilowii according to the Koch’s postulate. They were identified as Fusarium fujikuroi, F. proliferatum, Colletotrichum fructicola and C. liaoningense by combining morphological characteristics with phylogenetic analysis, with isolation frequencies of 31.4%, 8.6%, 20.0% and 40.0%, respectively. In addition, the indoor toxicity test results indicated that prochloraz, fludioxonil, difenoconazole, phenamacril and chlorothalonil had better inhibitory effect against F. fujikuroi and F. proliferatum, with EC₅₀ ranging from 0.1046 to 5.1781 μg/mL. Prochloraz, fludioxonil and difenoconazole had better inhibitory effect against C. fructiicola and C. liaoningense with EC₅₀ ranging from 0.0097 to 2.1325 μg/mL. Conclusion The causal agents associated with fruit rot disease on T. kirilowii are Fusarium spp. and Colletotrichum spp.. Prochloraz, fludioxonil and difenoconazole all show good inhibitory activity against the pathogens.