Five new chlamydosporol derivatives, named pleospyrones A-E (1-5), together with one known congener (6), were isolated from the culture of the endophytic fungus Pleosporales sp. Sigrf05, obtained from the medicinal plant Siraitia grosvenorii. The structures of the new compounds were elucidated mainly by analysis of the HRESIMS, and (1D, 2D) NMR data, while ECD and optical rotation calculations were used to assign the absolute configurations. The plausible biosynthetic pathway of these compounds were proposed. The isolated compounds were evaluated for their cytotoxicity, antifungal and antibacterial activities. Compounds 1, and 4-6 were cytotoxic against the tested cancer cells with IC50 values of 1.26~47.5 μM. Compounds 1-3 showed moderate antifungal activities against Magnaporthe oryzae, while compound 5 displayed weak antibacterial activity.