Biosurfactants are surface-active compounds that have sparked interest in recent years because of their environmental advantages over conventional surfactants. The aim of this study was to investigate the production of biosurfactants by soil fungi isolated from the Amazon forest. Fungi colonies were isolated from soil samples and screened for biosurfactant production in submerged fermentation. In addition, the influences of bioprocess factors (carbon source, nitrogen source, pH, and fermentation time) were investigated. Finally, the biosurfactant produced was semipurified and submitted to stability tests. One hundred fungal cultures were obtained from the soil samples, identified by micromorphology, and submitted to screening for biosurfactant production. Sixty-one strains produced biosurfactants. The strain Penicillium 8CC2 showed the highest emulsification index (54.2%). The optimized bioprocess conditions for biosurfactant production by Penicillium 8CC2 were as follows: soybean oil, 20 g/L; yeast extract, 30 g/L; pH 9; duration of 9 days. The semipurified biosurfactant showed stability after heating at 100°C for 60 min and after the addition of 30% NaCl (w/v). Tween 80 (0.2% w/v), a conventional surfactant, was used as the control.