The herbicide atrazine (2-chloro-4-ethylamine-6-isopropylamine-s-triazine) is extensively used for weed control in sugarcane crops. The application of fungi for the biodegradation of xenobiotics has been studied with promising results. Therefore, atrazine degradation mediated by Pleurotus ostreatus INCQS 40310 was evaluated, and the involvement of ligninolytic enzymes along with the degradation process was also investigated. To promote high degradation percentages and rates, a fractional factorial experimental design was first used to determine the most significant medium components for atrazine degradation. This strategy improved atrazine degradation from 39.0% to 71.0% after 15 days, with the formation of different metabolites. Afterward, a 3 2 full factorial design was performed using the variables selected in the first part of this study. The salts FeSO 4 and MnSO 4 showed significant influence in the percentages and the rates of atrazine degradation. The medium optimization resulted in 90.3% and 94.5% of atrazine degradation after 10 days and 15 days, respectively. Although laccase activity was measured during the degradation process, it was not possible to correlate laccase activity with atrazine degradation. The results demonstrated the efficiency of P. ostreatus INCQS 40310 for atrazine degradation, thus demonstrating the potential of this fungus as a bioremediation agent.