Anaerobic mesophilic co-digestion of mixed sewage sludge from wastewater treatment plants, WWTP, with crude glycerol, the major byproduct of the biodiesel industry, has been examined using a two-phase digestion process in a semi-continuous CSTR at laboratory scale. The objective was to determine the operational conditions that enhanced biohydrogen and methane production and to evaluate the effect of the organic loading rate (OLR) applied to the process. It was concluded that the Hydraulic Retention Time HRT of the methanogenic stage did not have an important influence on the operational process of co-digestion of sewage sludge and glycerol in terms of efficiency of organic removal and biogas yield. Hence, the results obtained were 73–77% organic matter removal (as CODr) with 0.032 LH2/gCODr and 0.16 LCH4/gCODr when the system operated at OLRs in the range of 15.33–17.90 gCODs/L d with HRTs of 3 days in the acidogenic digester and 6, 8, and 10 days in the methanogenic digester. In terms of volatile solids, the results obtained were 92–88% organic matter removal (as VSr) with 0.20 LH2/gVSr and 1.27 LCH4/gVSr when the system operated at OLRs in the range of 1.94–2.79 gVS/L d.However, the OLR had an important influence on the H2 and CH4 yields. Hence, the best operational condition was an OLR of 7.82 gCOD/L d, with removal of 93% CODr and hydrogen and methane yields of 0.026 LH2/gCODr and 0.29 LCH4/gCODr, respectively.In terms of volatile solids, the best operational condition was an OLR of 1.01 gVS/L d, with removal of 89% VSr and hydrogen and methane yields of 0.50 LH2/gVSr and 1.48 LCH4/gVSr, respectively.