Abstract Background The Brunei River and Bay estuarine system (BES) in the northwest of Borneo is acidic and highly turbid. The system supports extensive intertidal mudflats and presents a potentially steep salinity and pH gradient along its length (45 km). Temporal variation in physical parameters is observed diurnally due to seawater flux during tidal forcing, and stochastically due to elevated freshwater inflow after rains, resulting in a salinity range between 0 and 34 psu. High velocity freshwater run-off from acid sulphate formations during monsoon seasons results in highly variable and acidic conditions (pH 4) at the upper reaches of the BES, whereas the pH is relatively stable (pH 8) at the seaward extremes, due to mixing with seawater from the South China Sea. At their surfaces, the BES mudflats present microbial ecosystems driven by oxygenic phototrophs. To study the effect of various physical parameters on the bacterial diversity of the BES mudflats, surface samples were collected from six sites stretching over 40 km for molecular and phylogentic analysis. Results The bacterial diversity at these sites was compared by community fingerprinting analysis using 16S rRNA gene based denaturing gradient gel electrophoresis and by 16S rRNA gene sequencing and phylogenetic analyses. Results revealed functionally conserved, diatom-driven microbial mudflat communities composed of mainly novel, uncultured species. Species composition was evaluated as 50-70% unique for each site along the BES. Clustering of the sequences commonly occurred and revealed that proteobacterial diversity was related to the salinity gradient. When considering all phyla, the diversity varied consistently with physical parameters (including anthropogenic) that are expected to influence microbial composition. Conclusion The BES mudflats were found to comprise the typical functional groups of microorganisms associated with photosynthetic carbon flux, sulfur cycling (Gamma- and Deltaproteobacteria), and decomposition (Bacteroidetes). From a structural perspective, however, the mudflats constituted discretely distributed communities along the physical gradient of the BES, composed of largely novel species of Bacteria. This study provides first insights into patterns of bacterial community structure in tropical South East Asian coastal ecosystems that are potentially threatened by increasing variability in pH and salinity, in line with predicted future environmental change.