Biofilms play an important role as a settlement cue for invertebrate larvae and significantly contribute to the nutrient turnover in aquatic ecosystems. Nevertheless, little is known about how biofilm community structure generally responds to environmental changes. This study aimed to identify patterns of bacterial dynamics in coral reef biofilms in response to associated macrofouling community structure, microhabitat (exposed vs. sheltered), seasonality, and eutrophication. Settlement tiles were deployed at four reefs along a cross-shelf eutrophication gradient and were exchanged every 4 months over 20 months. The fouling community composition on the tiles was recorded and the bacterial community structure was assessed with the community fingerprinting technique Automated Ribosomal Intergenic Spacer Analysis (ARISA). Bacterial operational taxonomic unit (OTU) number was higher on exposed tiles, where the fouling community was homogenous and algae-dominated, than in sheltered habitats, which were occupied by a variety of filter feeders. Furthermore, OTU number was also highest in eutrophied near-shore reefs, while seasonal variations in community structure were most pronounced in the oligotrophic mid-shelf reef. In contrast, the macrofouling community structure did not change significantly with seasons. Changes in bacterial community patterns were mostly affected by microhabitat, seasonal and anthropogenically derived changes in nutrient availability, and to a lesser extent by changes in the macrofouling community structure. Path analysis revealed a complex interplay of various environmental and biological factors explaining the spatial and temporal variations in bacterial biofilm communities under natural conditions.