The combined use of a rhamnolipid biosurfactant produced by Pseudomonas aeruginosa and an enzyme pool produced by solid-state fermentation with Penicillium simplicissimum using babassu cake as culture medium in the anaerobic treatment of an effluent with a high fat content from a poultry processing plant was evaluated. Central composite rotatable design was used to evaluate the enzyme pool and biosurfactant concentrations and the treatment temperature of the effluent containing about 2400 mg oil and grease per liter. The combination that yielded the highest specific methane production was 0.19%(w/v) enzyme pool and 114 mg/L biosurfactant at 33 掳C. It could therefore be concluded that the combined use of a rhamnolipid biosurfactant with an enzyme preparation obtained from solid-state fermentation may enhance methane production and enable the use of anaerobic technology in this sector, eliminating the need for physicochemical processes or the addition of costly commercial enzymes.