In recent years, the use of agro-industrial residues as sources of biofuels has gained great attention worldwide due to limited reserves of fossil fuels. The sisal industry in Tanzania generates large quantities of sisal leaf decortications residues (SLDR) with good potential for bio-methanation. However, the process is limited by the Iignocellulosic nature of the SLDR. To improve methane production from the residue, pre-treatment is essential prior to anaerobic digestion. In this study, the effect of biological pre-treatment of SLDR with lignolytic and cellulolytic fungi using different inoculum concentrations and incubation periods, singly and in combination was investigated in anaerobic batch bioreactors at a volatile solids (VS) loading rate of 5.84 grams VS. Pre-treatment of SLDR for 4 days with strain CCHT-1 and for 8 days with Trichoderma reesei at inoculum concentrations of 10% and 25% separately, gave methane yields of 0.203 m3 CH4/kg VSadded and 0.192 m3 Cfy/kg VS added, respectively. In a two-steps pre-treatment using the two organisms in succession, first using strain CCHT-1 followed by T. reesei, an increase of about 101% in methane yield was obtained. On the other hand using T. reesei first followed by strain CCHT-1, the methane yield increase dropped to 30%. Within the experimental conditions, the results confirmed that biological pre-treatment has the potential to achieve significant improvement in biogas production from SLDR in a two-steps pre-treatment approach using CCHT-1 followed by T. reesei. It was concluded that this large reservoir of biomass from the sisal industry could be harnessed for methane production by sisal decortication factories.