Nanoparticles (NPs) and nanomaterials (NMs) are now widely used in a variety of applications, including medicine, solar energy, drug delivery, water treatment, and pollution detection. Hematite (α-Fe2O3) nanoparticles (Hem-NPs) were manufactured in this work by utilizing a cost-effective and ecofriendly approach that included a biomass filtrate of A. niger AH1 as a bio-reducer. The structural and optical properties of Hem-NPs were investigated using X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), and UV-visible and Fourier-transform infrared (FTIR) spectroscopies. The results revealed that all of the studied parameters, as well as their interactions, had a significant impact on the crystallite size. The average diameter size of the biosynthesized Hem-NPs ranged between 60 and 80 nm. The antimicrobial and photocatalytic activities of Hem-NPs were investigated. The antimicrobial results of Hem-NPs revealed that Hem-NPs exhibited antibacterial activity against E. coli, B. subtilis, and S. mutans with MICs of 125, 31.25, and 15.62 µg/mL, respectively. Moreover, Hem-NPs exhibited antifungal activity against C. albicans and A. fumigatus, where the MICs were 2000 and 62.5 µg/mL, respectively. The efficiency of biosynthesized Hem-NPs was determined for the rapid biodegradation of crystal violet (CV) dye, reaching up to 97 percent after 150 min. Furthermore, Hem-NPs were successfully used more than once for biodegradation and that was regarded as its efficacy. In conclusion, Hem-NPs were successfully biosynthesized using A. niger AH1 and demonstrated both antimicrobial activity and photocatalytic activity against CV dye.