A strain of Penicillium corylophilum isolated from Brazilian soil sample was submitted to different culture conditions to investigate the production of secondary metabolites with antimicrobial activity. The largest number of conidia was obtained after 5 days of incubation in oat medium and the highest level of antimicrobial activity was produced when the fungus culture was developed in the Czapek medium. The activity against Staphylococcus aureus was found only in the chloroform extract from Czapek culture broth, which also showed activity against Micrococcus luteus. Fumiquinozoline F was isolated from the active chloroform extract by using chromatographic methods. The minimal inhibitory concentration (MIC) values for M. luteus and S. aureus were 99 microg/ mL and 137microg/mL, respectively.