At deep-sea hydrothermal vents, reduced, super-heated hydrothermal fluids mix with cold, oxygenated seawater. This creates temperature and chemical gradients that support chemosynthetic primary production and a biomass-rich community of invertebrates. In late 2005/early 2006 an eruption occurred on the East Pacific Rise at 9°50′N, 104°17′W. Direct observations of the post-eruptive diffuse-flow vents indicated that the earliest colonizers were microbial biofilms. Two cruises in 2006 and 2007 allowed us to monitor and sample the early steps of ecosystem recovery. The main objective of this work was to characterize the composition of microbial biofilms in relation to the temperature and chemistry of the hydrothermal fluids and the observed patterns of megafaunal colonization. The area selected for this study had local seafloor habitats of active diffuse flow (in-flow) interrupted by adjacent habitats with no apparent expulsion of hydrothermal fluids (no-flow). The in-flow habitats were characterized by higher temperatures (1.6–25.2 °C) and H2S concentrations (up to 67.3 µM) than the no-flow habitats, and the microbial biofilms were dominated by chemosynthetic Epsilonproteobacteria. The no-flow habitats had much lower temperatures (1.2–5.2 °C) and H2S concentrations (0.3–2.9 µM), and Gammaproteobacteria dominated the biofilms. Siboglinid tubeworms colonized only in-flow habitats, while they were absent at the no-flow areas, suggesting a correlation between siboglinid tubeworm colonization, active hydrothermal flow, and the composition of chemosynthetic microbial biofilms.