In the healthcare sector, the production of bioactive silver nanoparticles (AgNPs) with antimicrobial properties is of great importance. In this study, a novel bacterial strain, Paenibacillus sp. MAHUQ-63, was identified as a potential candidate for facile and rapid biosynthesis of AgNPs. The synthesized AgNPs were used to control the growth of human pathogens, Salmonella Enteritidis and Candida albicans. The bacterial culture supernatant was utilized to synthesize the nanoparticles. FE-TEM examination showed spherical-shaped nanoparticles with 15 to 55 nm in size. FTIR analysis identified various functional groups. The synthesized AgNPs demonstrated remarkable activity against S. Enteritidis and C. albicans. The zone of inhibition (ZOI) for 100 µL (0.5 mg/mL) of AgNPs against S. Enteritidis and Candida albicans were 18.0 ± 1.0 and 19.5 ± 1.3 mm, respectively. The minimum inhibitory concentrations (MICs) were 25.0 and 12.5 μg/mL against S. Enteritidis and Candida albicans, respectively. Additionally, the minimum bactericidal concentrations (MBC) were 25.0 μg/mL against both pathogenic microbes. The FE-SEM analysis showed that the treatment of AgNPs caused morphological and structural damage to both S. Enteritidis and Candida albicans. Therefore, these AgNPs can be used as a new and effective antimicrobial agent.