Agave plants grow in semi-arid regions and are used for mescal production. However, agave fiber by-products are considered waste materials. Thus, we tested agave fiber as a filter media and biofilm material carrier for removing pollutants from municipal wastewater. Three laboratory-scale biofiltration reactors were used in two trials with five hydraulic loading rates (HLRs = 0.27, 0.54, 0.80, 1.07 and 1.34 m3 m−2 d−1). One series was conducted using mechanical aeration (0.62 m3 m−2 h−1). To prevent compaction, decreasing pressure and clogging of the filter media, 4, 8 and 12 internal divisions were evaluated in the biofilter column. After 17 months of continuous operation at an HLR of 0.80 m3 m−2 d−1, the removal efficiencies of the aerated biofilters were 92.0% biochemical oxygen demand, 79.7% chemical oxygen demand, 98.0% helminth eggs, 99.9% fecal coliforms and 91.9% total suspended solids. Statistical analysis showed that the chosen operational parameters significantly influenced the removal efficiencies of the biofilters. The effluent quality obtained under these conditions complied with the Mexican and US EPA standards for agricultural irrigation and green spaces, except for coliforms, which is why the effluents must be disinfected. Thus, agave fiber is a favorable choice for use as a packing material in biofiltration processes.