Fauna from deep-sea cold seeps worldwide is dominated by chemosymbiotic metazoans. Recently, investigation of new sites in the Gulf of Guinea yielded numerous new species for which symbiosis was strongly suspected. In this study, symbioses are characterized in five seep-specialist metazoans recently collected from the Guiness site located at ∼600 m depth. Four bivalve and one annelid species belonging to families previously documented to harbor chemosynthetic bacteria were investigated using bacterial marker gene sequencing, fluorescence in situ hybridization, and stable isotope analyses. Results support that all five species display chemosynthetic, sulfur-oxidizing γ-proteobacteria. Bacteria are abundant in the gills of bivalves, and in the trophosome of the siboglinid annelid. As observed for their relatives occurring at deeper sites, chemoautotrophy is a major source of carbon for animal nutrition. Although symbionts found in each host species are related to symbionts found in other metazoans from the same families, several incongruencies are observed among phylogenetic trees obtained from the different bacterial genes, suggesting a certain level of heterogeneity in symbiont strains present. Results provide new insights into the diversity, biogeography, and role of symbiotic bacteria in metazoans from the Gulf of Guinea, at a site located at an intermediate depth between the continental shelf and the deep sea.