Abstract: The main objective of this study was to characterize metagenomically and evaluate the biofilter effectiveness for COD, total coliforms, Escherichia coli, and Helminth eggs removal from urban wastewater using different configurations. Several contact times of wastewater with the biofilm were tested, as well as physicochemical and biological parameters. The optimal treatment time for COD removal was 480 min for BAC1 and 360min for BAC2, while for E. coli removal, it was 90 and 120 min by BAC1 and BAC2, respectively, and 200 min for Helminth eggs by both BACs. The metagenomic analysis revealed a diverse microbial community within the biofilms, which contributed to the biodegradation process. BAC1 exhibited greater microbiological diversity and better performance than BAC2. Key microbial species identified, such as Lysinibacillus sphaericus, Citrobacter freundii, and Saccharomyces sp. possess notable biodegradative and antimicrobial properties, further enhancing the filter’sefficacy. The effluents treated by both BACs did not exhibit toxicity for germination and root elongation of radish seeds Ver menos