Analyses of biofilm community structure may potentially be employed for aquatic ecosystem health assessment, however, to date, biofilm diversity within urban lakes using reclaimed water has not been examined. Accordingly, the microbial community diversity and structure of biofilms from the surface of multiple matrices with varying roughness (0.1, 1.0 and 10.0 μm) were characterized using a suite of molecular techniques including scanning electron microscopy, genetic fingerprinting and phospholipid-derived fatty acid analyses. Samples were largely comprised of inorganic particles, algae and numerous bacterial species; 12 phospholipid-derived fatty acid (PLFA) types were identified, significantly less than typically associated with sewage. Both growth matrix surface roughness and biofilm growth phase were shown to concur with significantly different microbial quantity and community structures. Gram-negative bacteria bacillus i15:03OH and 18:0 were the dominant bacterial genera, collectively comprising ≈75 % of identified PLFA species content. Calculated species diversity (H) and species dominance (D) exhibited identical correlational patterns with measured water quality parameters; significant positive correlations were exhibited with respect to Mg2, while significant negative correlations were found for NO3, TP, BOD, COD, SP, PO4, SO4 and pH. Results indicate that analyses of biofilm formation and structure could be effectively used to undertake integrated assessments of the ecological health of lake systems using reclaimed water. Further work is required to elucidate the optimum conditions for sample collection and analytical interpretation