Since silver nanoparticles (AgNPs) have antiplasmodial, antibacterial, and antifungal properties, they offer promise as a therapeutic tool for the treatment of various illnesses and parasites. AgNO3 solution was employed in conjunction with cell-free supernatant from Levilactobacillus brevis cultivated on MRS medium to synthesize silver nanoparticles. Using the well diffusion method, samples' antimicrobial activity was evaluated in vitro against a variety of pathogens, such as filamentous fungi Aspergillus brasiliensis ATCC 16404, unicellular fungi Candida albicans ATCC 10231, and Gram positive bacteria Bacillus spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, and Gram negative bacteria Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 8739. Furthermore, the minimum inhibitory concentration (MIC) of AgNPs against Staphylococcus aureus ATCC 6538 was determined using a range of concentrations (0, 5, 10, 15, 20, 25, 50, 75, and 100 µg). The growth of both Gram positive and Gram negative bacteria was inhibited by 100 µg AgNPs. It has been observed that filamentous and unicellular fungi are resistant to AgNPs. The MIC for 20 µg of AgNPs was found from the results. Given these encouraging therapeutic properties, the use of AgNPs in the control of infectious diseases is warranted.