The use of microorganisms to treat effluents can reduce problems caused by oils and fats in subsequent treatment stages. The present work investigates the use of a lipase-producing microorganism in the pretreatment of effluent from a fish slaughterhouse. Initial preliminary tests were performed with two microorganisms: Staphylococcus warneri and Bacillus megaterium. The following parameters were evaluated after the treatment: chemical oxygen demand (COD), oils and greases (O&G), and lipolytic activity. Bacillus megaterium provided the highest removal efficiencies for the analyzed parameters, consequently this microorganism was then used for the pretreatment of fish slaughterhouse effluent. A central composite rotational design (CCRD) was applied, with pH, temperature, and inoculum concentration as the independent variables. The response variables were lipolytic activity, COD, O&G, free fatty acids, and pH, for a reaction time of 72 h. The maximum values obtained after the treatment were lipolytic activity of 0.25 U mL− 1, COD and O&G removals of 61.11% and 85.8%, respectively, and free fatty acids production of 12.54 µmol mL− 1. The findings showed that the application of Bacillus megaterium significantly reduced the levels of oils and fats, demonstrating the potential of the enzyme for treatment of this type of effluent.