The effect of different hydrolysis times (4, 8 and 24 h) of a lipase-rich enzymatic preparation (0.1, 0.5 and 1.0% w/v) produced by fungus Penicillium sp. in solid-state fermentation was evaluated on the anaerobic biodegradability of a poultry slaughterhouse wastewater with 800 mg oil and grease [O&G]/L in three sequential batches. The enzymatic pre-treatment of O&G in the experiments with no acclimated sludge had no discernible effect because regardless of the conditions adopted COD removal efficiencies of 95.3% to 98.7% were obtained. However, when the sludge was reused (once or twice) the COD removal efficiencies in the control experiments (69.8% and 53.4%) were considerably lower than in the experiments with hydrolyzed effluent (of 93.8% to 98.4%). Higher values of specific methane production were obtained with 0.1% SEP and 4 h of hydrolysis. After acclimation of the sludge, 19.9% fat was still adhered to the sludge in the control experiment, while the sludge in the experiment with enzymatically pre-treated effluent contained only 8.6% fat, confirming the accumulation of fat when the enzyme pool was not used.