Abstract This study reports the use of statistical mixture design as a tool for the simultaneous production of lipase, CMCase, α-amylase, and β-glucosidase by Aspergillus niger under solid-state fermentation. Wheat bran, soybean meal, cottonseed meal, and orange peel were used as substrates, either individually or combined in different formulations, to study their synergistic or antagonistic effects on production of the enzymes. The highest lipase (323 U g-1) and CMCase (10 U g-1) activities were detected after 48 h, while the maximum activities of α-amylase (18 U g-1) and β-glucosidase (15 U g-1) occurred at 72 and 96 h, respectively. Considering the substrate formulation, the ternary mixture of wheat bran (1/3), soybean meal (1/3), and cottonseed meal (1/3) was the most versatile, showing production of CMCase (>5 U g-1) and α-amylase (>8 U g-1) at 24 h, lipase (>320 U g-1) at 72 h, and β-glucosidase (>10 U g-1) at 48 h.