Currently, nanoparticles and nanomaterials are widely used for biomedical applications. In the present study, silver nanoparticles (AgNPs) were successfully biosynthesized using a cell-free extract (CFE) of Bacillus thuringiensis MAE 6 through a green and ecofriendly method. The size of the biosynthesized AgNPs was 32.7 nm, and their crystalline nature was confirmed by XRD, according to characterization results. A surface plasmon resonance spectrum of AgNPs was obtained at 420 nm. Nanoparticles were further characterized using DLS and FTIR analyses, which provided information on their size, stability, and functional groups. AgNPs revealed less cytotoxicity against normal Vero cell line [IC50 = 155 μg/mL]. Moreover, the biosynthesized AgNPs exhibited promising antifungal activity against four most common Aspergillus, including Aspergillus niger, A. terreus, A. flavus, and A. fumigatus at concentrations of 500 μg/mL where inhibition zones were 16, 20, 26, and 19 mm, respectively. In addition, MICs of AgNPs against A. niger, A. terreus, A. flavus, and A. fumigatus were 125, 62.5, 15.62, and 62.5 μg/mL, respectively. Furthermore, the ultrastructural study confirmed the antifungal effect of AgNPs, where the cell wall’s integrity and homogeneity were lost; the cell membrane had separated from the cell wall and had intruded into the cytoplasm. In conclusion, the biosynthesized AgNPs using a CFE of B. thuringiensis can be used as a promising antifungal agent against Aspergillus species causing Aspergillosis.