Two- and single-stage anaerobic treatment systems were assessed for treatment performance and for bioenergy production from sucrose-based wastewater. In the two-stage system, a hydrogen-producing upflow anaerobic sludge blanket reactor (HU reactor) was used in the acidogenic phase. The methanogenic reactor of the two-stage system (MF reactor) and the single-stage reactor (SSF reactor) were structured fixed-bed reactors. The two-stage system showed superior performance, evidenced by lower organic acids, chemical oxygen demand (COD) and suspended solids concentrations in the effluent, and higher biogas methane content and yield. Continuous and stable H2 production was obtained in the acidogenic reactor. At the end of operation, the organic loading rates applied to the two- and single-stage systems were 6.4 and 5.2 gCOD L−1 d−1, respectively. Under these conditions, the effluent soluble COD and volatile suspended solids (VSS) concentrations were 165 and 92 mg L−1 in the two-stage system, and 256 and 244 mg L−1 in the single-stage system, respectively. The energy yield of the two-stage system was 20.69 kJ g−1CODadded, which was 34% higher than the yield of the single-stage system. The sequencing analyses showed that the archaeal distribution changed little between the inoculum and sludge from the MF reactor, in which acetoclastic Methanosaeta was predominant. However, hydrogenotrophic Methanospirillum was found most, followed by Methanosaeta, in the sludge from the SSF reactor.