The bioconversion of porungo cheese whey to produce β-galactosidase in batch system was studied. The whey released after curd cutting and precipitation during porungo cheese production was collected in borosilicate flasks. Two strains of Kluyveromyces marxianus, CCT 4086 and CBS 6556, and whey supplementation with different nitrogen sources were evaluated. Different temperatures (30 °C and 37 °C) and pH values (5.0 to 7.0) were investigated to establish the best conditions for enzyme production. The highest enzymatic activity was obtained by K. marxianus CCT 4086 in porungo cheese whey supplemented with yeast extract (16.73 U mL-1). K. marxianus CCT 4086 produced superior β-galactosidase activity when compared to CBS 6556 for all media tested (ranging from 11.69 to 14.40 U mL-1). Highest β-galactosidase activity was reached under conditions of pH 7.0 and 30 °C using K. marxianus CCT 4086 in the better media composition. The lowest enzymatic activity was observed at 37 °C for all pH values tested (10.69 U mL-1 to 13.94 U mL-1) and a highest β-galactosidase activity was reached in pH 7.0 for both two temperatures (11.42 to 15.93 U mL-1). Porungo cheese whey shows potential for industrial β-galactosidase production by microbial fermentation.