Different types of carriers were tested as support material in a lab-scale moving bed biofilm reactor (MBBR) used to treat urban wastewater under three different conditions of hydraulic retention time (HRT) and carrier filling ratios (FR). The bacterial diversity developed on the biofilms responsible of the treatment was studied using a cultivation-independent approach based on the polymerase chain reaction-temperature gradient gel electrophoresis technique (PCR-TGGE). Cluster analysis of TGGE fingerprints showed significant differences of community structure dependent upon the different operational conditions applied. Redundancy analysis (RDA) was used to determine the relationship between the operational conditions (type of carrier, HRT, FR) and bacterial biofilm diversity, demonstrating a significant effect of FR = 50%. Phylogenetic analysis of PCR-reamplified and sequenced TGGE bands revealed that the prevalent Bacteria populations in the biofilm were related to Betaproteobacteria (46%), Firmicutes (34%),Alphaproteobacteria (14%) and Gammaproteobacteria (9%).