Combination therapies targeting multiple organs and metabolic pathways are promising therapeutic options to combat obesity progression and/or its comorbidities. The alterations in the composition of the gut microbiota initially observed in obesity have been extended recently to functional alterations. Bacterial functions involve metabolite synthesis that may contribute to both the gut microbiota and the host physiology. Among them are B vitamins, whose metabolism at the systemic, tissue, or microbial level is dysfunctional in obesity. We previously reported that the combination of oral supplementation of a prebiotic (fructo-oligosaccharides, FOS) and vitamin B7/B8 (biotin) impedes fat mass accumulation and hyperglycemia in mice with established obesity. This was associated with an attenuation of dysbiosis with improved microbial vitamin metabolism. We now extend this study by characterizing whole body energy metabolism along with adipose tissue transcriptome and histology in this mouse model. We observed that FOS resulted in increased caloric excretion in parallel with downregulation of genes and proteins involved in jejunal lipid transport. The combined treatments also strongly inhibited the accumulation of subcutaneous fat mass, with a reduced adipocyte size and expression of lipid metabolism genes. Downregulation of inflammatory and fibrotic genes and proteins was also observed in both visceral and brown adipose tissues and liver by combined FOS and biotin supplementation. In conclusion, oral administration of a prebiotic and biotin has a beneficial impact on the metabolism of key organs involved in the pathophysiology of obesity, which could have promising translational applications.