The use of a biosurfactant surfactin produced by Bacillus subtilis LB5a in cassava growth medium (cassava wastewater) was evaluated to treat anaerobically, the effluent from a poultry slaughterhouse. During the effluent pretreatment, effects of surfactin concentration factors were evaluated, considering the ones which were superior and below its critical micelle concentration (CMC = 28 mg L-1): 6, 13, 27, 31, 48, 73 and 56 mg L-1 and temperature (25, 30, 42.5, 55 and 60°C) up to 6 h, using the rotational central composite design. During anaerobic treatment, flasks were filled with anaerobic sludge as inoculum and a pretreated effluent for 4.5 h in its different concentrations of surfactin. Reactors were connected to eudiometers under static system at 34°C. During the pretreatment phase, there was a direct ratio between temperature and surfactin concentrations according to the increase of organic matter solubilization, measured by soluble chemical oxygen demand (SCOD). These results have shown surfactin applicability produced by B. subtilis LB5a during the anaerobic treatment of effluent from a poultry slaughterhouse; as for all treatments, there was no inhibition of microbial consortium of the anaerobic sludge. SCOD removal was above 80%, while oil and greases removal was above 70%, plus a propitious specific methane yield. Key words: Bacillus subtilis, agro-industrial effluents, anaerobic sludge.