This study qualitatively assessed the impacts of different start-up strategies on the performance of methane (CH4) production from cattle manure (CM) in electromethanogenic reactors. Single chamber MECs were operated with an applied voltage of 0.7 V and the impact of electrode acclimatization with a simple substrate, acetate (ACE) vs a complex waste, CM, was compared. Upon biofilm formation on the sole carbon source (ACE or CM), several MECs (ACE_CM and CM_ACE) were subjected to cross-feeding (switching substrate to CM or ACE) during the test period to evaluate the impact of the primary substrate. Even though there was twice as much peak current density via feeding ACE during biofilm formation, this did not translate into higher CH4 production during the test period, when reactors were fed with CM. Higher or similar CH4 production was recorded in CM_CM reactors compared to ACE_CM at various soluble chemical oxygen demand (sCOD) concentrations. Additionally, feeding ACE as primary substrate did not significantly impact either COD removals or coulombic efficiencies. On the other hand, the use of anaerobic digester (AD) seed as an inoculum in CM-fed MECs (CM_CM), relative to no inoculum added MECs (Blank), increased the initial CH4 production rate by 45% and reduced the start-up time by 20%. In CM-fed MECs, Geobacter dominated bacterial communities of bioanodes and hydrogenotrophic methanogen Methanoculleus dominated archaeal communities of biocathodes. Community cluster analysis revealed the significance of primary substrate in shaping electrode biofilm; thus, it should be carefully selected for successful start-up of electromethanogenic reactors treating wastes.