Pectinase production by Aspergillus niger LB-02-SF was studied in a bench-scale rotating drum bioreactor. With 30% of the bioreactor volume occupied, agitation promoted increasing fungal growth and decreasing enzyme production, whereas with 50% occupation, an opposite trend occurred. The interval between agitation events was evaluated for an occupation volume of 70%, and no difference in enzyme production was observed for the 90 and 120 min intervals; for the 60 min intervals, however, the activity was reduced. Cultivations performed without temperature control attained 45 °C and showed maximum enzyme activity almost four times superior to cultivations controlled at 30–37 °C. Air flow rates between 0.18 and 0.90 L of air per kilogram of medium per minute were evaluated for 30 and 50% of the occupation volume. Fungal growth and pectinase production were favoured by the smallest and largest occupation volumes, respectively, with an intermediate air flow. The results of this work indicate that the best conditions for biomass growth are not the best for the production of pectinases.