Abstract Aspergillus species have been used in different industrial applications including extracellular enzyme production and as potential hosts for recombinant heterologous proteins production because of their excellent growth and fermentation properties. However, recombinant protein production levels are typically low due to the impact of Aspergillus proteases in modifying and degrading the heterologous proteins, especially in the extracellular medium. The potential of using the protease inhibitor pepstatin in filamentous Aspergillus niger culture was investigated in this study. The majority of protease activity was released into the medium as biomass content decreases after maximum biomass concentration was attained and a low concentration of pepstatin effectively inhibited the extracellular protease activity to an extent of 80–90%. The effectiveness of pepstatin in inhibiting the majority of protease activity was further demonstrated in media supplemented with both non-peptide and peptide-containing nitrogen sources. Pepstatin addition had no effect on microbial growth. Dilution of the culture filtrate released inhibition, indicating that pepstatin did not have a notable effect on protease production and that inhibition was reversible.