In order to develop natural antibacterial agents, the antibacterial activity of Aspergillusnigerxj was investigated. After being cultured in potato dextrose liquid medium liquid medium, mycelia was under heating reflux extraction with 90% ethanol. Removal of ethanol under reduced pressure gave a residue, to which water was added and then extracted with petroleum ether and ethyl acetate. In vacuo evaporation of the solvents yielded three crude extracts. Then the disc diffusion method was used to measure the antibacterial activity of these extracts. The petroleum ether extract with antibacterial activity was separated by silica gel column chromatography method, then separated and identified by GC-MS after been methyl esterified. At the concentration of 50 mg/mL, the petroleum ether extract of mycelia exhibited inhibitory effect against Staphylococcus aureus. The petroleum ether extract from Aspergillusnigerxj mycelia contained natural substances with antibacterial activity and fatty acids are the main constituents in it.