Acid Orange 24 (AO24) dye was degraded in a trickling biofilter packed with peat and wood chips and inoculated with biomass from a petrochemical industry wastewater system. Different operating strategies were tested; in the first stage, two biofilters were operated independently--one non-aerated biofilter (passive) and the other with aeration-subsequently, the systems were operated serially, and effluent from the non-aerated biofilter was fed to the biofilter with aeration. This treatment train was used to test three different filtration velocities--0.141, 0.282, and 0.423 m/d. The results show that, when operating the systems with a dye charge of 0.035 kg AO24 m2/d and treating the effluent in a single step, good removal efficiencies of AO24 (95 and 89%), COD (63 and 53%), and acute toxicity (63 and 78%) were obtained in both biofilters (with and without air), although mutagenic and potentially carcinogenic intermediary compounds were not removed, because genotoxicity exhibits values higher than 2.0 units for the mutation rate. When using the non-aerated biofilter/aerated biofilter treatment train, it is possible to treat a dye charge 3 times greater (0.106 kg AO24 m2/d) and efficiently remove 98% AO24, 76% COD, 100% acute toxicity, and 100% genotoxicity, which indicates that, with this biological system, an advanced degree of biotransformation and mineralization of the azo dye AO24 is achieved.