No doubt that studying the secondary metabolites produced by plants can lead to the detection of new pesticides. In this study, we followed the bioassay-guided method to isolate active ingredients from Periploca sepium root barks (PSRB) and verified their antifungal activity against seven phytopathogenic fungi in vitro. A total of six pregnane glycosides were isolated and identified as periplocoside A (PSA), periplocoside D (PSD), periplocoside E (PSE), periplocoside F (PSF), periplocoside N (PSN) and periploside W (PSW). Bioassay results revealed that PSW exhibited highly significant antifungal activity against Valsa mali and Fusarium graminearum with EC50 values of 26.50 mg/L and 93.94 mg/L, respectively. In controlling apple Valsa canker, PSW (1000 mg/L) exhibited significantly higher protective activity with 85.40% than that efficacy with 64.62% obtained by carbendazim (200 mg/L). Whereas there is no significant difference between both of them in the curative efficacy with 71.32% and 69.70%, respectively. In conclusion, our study recommended the pregnane glycosides from PSRB as a new class of fungicides or fungicide lead compounds not only because of their potential antifungal activity, but also because of their safety for human beings, mammals and non-target organisms.