Sulfide ‘chimney’ structures characteristic of seafloor hydrothermal activity are extreme microbial habitats. 13C/12C ratios of microbial membrane lipids have rarely been used to investigate bacterial and archaeal carbon assimilation pathways on these structures, despite complementing existing gene- and culture-based investigations. Here, we integrate analysis of IPL diversity and δ13C values (δ13Clipid) with 16S rRNA gene based taxonomy to examine microbial carbon flow patterns on active and inactive chimney structures from the Manus Basin. Surficial crusts of active structures, dominated by epsilonproteobacterial communities, all yield bacterial δ13Clipid values higher than biomass (total organic carbon) δ13C, implicating CO2 fixation via the reverse tricarboxylic acid cycle. Our data also suggest δ13Clipid values vary on individual active structures without accompanying changes in microbial diversity. Temperature and/or dissolved substrate effects on 13C fractionation during assimilation may be responsible, likely due to variable advective/diffusive fluid fluxes to chimney exteriors. In an inactive structure, δ13Clipid values lower than biomass and a distinctive IPL and 16S rRNA gene diversity suggest a shift to a more diverse community and an alternate carbon assimilation pathway after venting ceases. We discuss here the potential of IPL and δ13Clipid analyses to elucidate microbial carbon flow in hydrothermal structures when combined with other molecular tools.