To study the chemical components and biological activity of the endophytic fungus Nigrospora sp. from Panax notoginseng, various chromatographic separation techniques, including silica gel column chromatography and high-performance liquid chromatography, were employed. Three polyketide compounds were isolated from the ethyl acetate extract of the potato liquid fermentation products of this strain. Their structures were determined using methods such as NMR, HR-ESI-MS, and ECD, and were identified as (R)-2-methyl-4-(2-oxobutyl)cyclopent-2-en-1-one (1), multiplolide A (2), and 8-O-acetylmultiplolide (3). Among them, compound 1 was a new cyclopentenone derivative, while compounds 2 and 3 were derivatives of ten-membered macrolides, all of which were isolated from Nigrospora sp. for the first time. Antibacterial activity results indicated that compound 1 exhibited weak antibacterial activity against Escherichia coli, with a minimum inhibitory concentration of 64 µg/mL.