The fungal pathogen(TXLHFJ-2) causing brown rot fruit of Shengzhou nane was isolated and purified by tissue isolation method from the diseased fruits. The pathogen was identified as Monilinia fructicola by morphological characteristics and polygenic joint sequence analysis including fragment of nuclear ribosomal internal transcribed spacer region(ITS), translation elongation factor-1(TEF-1α) and β-tubulin. Further observation revealed the optimum temperature for Monilinia fructicola growth was 22.5 ℃. Six fungicides added to PDA medium respectively were screened indoors to evaluate the control effect on Monilinia fructicola, and the results showed that 5.0 mg/L tebuconazole, 5.0 mg/L thiophanate methyl, 5.0 mg/L iprodione, 25.0 mg/L difenoconazole, 90.0 mg/L boscalid and 100.0 mg/L zineb could completely inhibit the mycelia growth. In order to clarify the antifungal mechanism, Janus green B, Mito-Tracker Red CMXRos and Propidium bromide(PI) solution were used to stain mycelia after fungicides treatments. The results showed that compared to the control, fluorescence intensity in mycelia stained by Janus green B, Mito-Tracker Red CMXRos was obviously weaker, while red fluorescence intensity stained by PI was stronger in fungicide treated group. The above results showed that six fungicides could destroy the cell membrane integrity of Monilinia fructicola, and reduce the formation of mitochondrial membrane potential, thereby inhibit the pathogen growth. Field experiment indicated that thiophanate methyl, tebuconazole, and difenoconazole showed excellent antifugal efficacy, with 81.63%, 80.49%, and 79.98%, respectively. However, based on pesticide management measures of China, difenoconazole was recommended as the suitable fungicide for controlling Monilinia fructicola causing brown rot in Shenzhou nane fruits in practice.