This study aimed to better understand the role of different microbial groups and the determining fermentation pathways in a biohydrogen (bioH(2))-producing reactor fed with sugarcane vinasse by using next-generation sequencing and principal component analysis (PCA). Both microbial structure and dynamics were characterized. The highest bioH(2) production levels were associated with the Thermoanaerobacterium genus, whilst lactate-producing bacteria comprised the dominant genera (e.g. Lactobacillus and Leuconostoc) within the reactor at both stable and unstable bioH(2)-producing periods. PCA further revealed that the fermentation of lactate played a dual role in the reactor, as both bioH(2)-producing (acetate + lactate -> butyrate + bioH(2)) and non-bioH(2)-producing (lactate. propionate + acetate) routes could be observed. Overall, the results suggested that lactate is the primary alternative carbon source in vinasse-fed systems subjected to carbohydrate-shortage conditions.