At hydrothermal vent sites, chimneys consisting of sulfides, sulfates, and oxides are formed upon contact of reduced hydrothermal fluids with oxygenated seawater. The walls and surfaces of these chimneys are an important habitat for vent-associated microorganisms. We used community proteogenomics to investigate and compare the composition and in situ protein expression of microbial communities colonizing two actively venting hydrothermal chimneys from the Manus Basin back-arc spreading center (Papua New Guinea).