Resumo. The micro-organisms represent the greatest biological diversity, chemistry, molecular and physiological ofliving and isolation from natural resources such as soil, is of great importance for obtaining new strains.The objective of this study was to isolate and select strains of fungi with potential for the productionof β-galactosidase, with transgalactosylation activity. Were collected soil samples, serial dilutions andinoculated on Sabouraud ágar modified with lactose. The fungi were transferred to obtain isolatedcolonies and selected micro-organisms were identified by cultivation between slide and coverslip. Thefermentation process for production of the enzyme was made solid state with lactose (1% w/v) in cultureflasks and maintained at 26 °C for 7 days. The β-galactosidase produced was partially purified withethanol and determined the effects of pH and temperature on enzyme activity through two-level factorialdesign. The transgalactosylation activity was assessed by varying the pH and enzyme concentration andprofile of carbohydrates obtained was analyzed by high performance liquid chromatography. The fungusthat expressed the highest activity of β-galactosidase was identified as Penicillium sp and presented for theenzymatic activity optimum values of pH and temperature of 4.5 and 60 °C, respectively. The β-galactosidaseproduced by the fungus Penicillium sp presented hydrolytic activity and transgalactosylation having thepotential to be exploited industrially in the production of prebiotic.