Enzymatic hydrolysis of cellulose-containing raw materials, using Aspergillus niger, were studied. Filter paper, secondary cellulose-containing or starch-containing raw materials, miscanthus cellulose after alkaline or acid pretreatment, and wood chip cellulose, were used as substrates. The study focused on a wild A. niger strain, treated, or not (control), by ultraviolet (UV) irradiations for 45, 60, or 120 min (UV45, UV60, or UV120), or by UV irradiation for 120 min followed by a chemical treatment with NaN3 + ItBr for 30 min or 80 min (UV120 + CH30 or UV120 + CH80). A mixture of all the A. niger strains (MIX) was also tested. A citrate buffer, at 50 mM, wasthe most suitable for enzymatic hydrolysis. As the UV exposure time increased to 2 h, the cellulase activity of the surviving culturewas increased (r = 0.706; p < 0.05). The enzymatic activities of the obtained strains, towards miscanthus cellulose, wood chips, and filter paper, were inferior to those obtained with commercial enzymes (8.6 versus 9.1 IU), in some cases. Under stationary hydrolysis at 37 °C, pH = 4.7, the enzymatic activity of A. niger UV120 + CH30 was 24.9 IU. The enzymatic hydrolysis of secondary raw materials, using treated A. niger strains, was themost effective at 37 °C. Similarly, the most effective treatment of miscanthus cellulose and wood chips occurred at 50 °C. The maximum conversion of cellulose to glucose was observed using miscanthus cellulose (with alkaline pretreatment), and the minimum conversion was observed when using wood chips. The greatest value of cellulase activity was evidenced in the starch-containing raw materials, indicating that A. niger can ferment not only through cellulase activity, but also via an amylolytic one.