Atrazine is a commonly used herbicide in the Mexican agricultural industry even though it is hazardous for human and animal health. Before reaching its mineralization, this herbicide infiltrates through soil, producing toxic compounds and contaminating groundwater sources. Considering that Aspergillus niger (A. niger) is a filamentous fungus that degrades pectin, cellulose, and other organic polymers, this study analyzes its use to accelerate atrazine mineralization under optimal parameters of temperature, soil humidity, and a co-substrate for bio-stimulation. Different atrazine concentrations were used, from 0 ppm to 1000 ppm, in order to measure the growth of its filaments. Glucose and Opuntia ficus indica residues were analyzed as co-substrates for A. niger biodegradation improvement. A DOE analysis was used to establish optimal treatment conditions. The findings in this study show that a 10 wt% of O. ficus indica as a co-substrate with 80% humidity achieved atrazine biodegradation in the order of 70% to 75% in 6 days.