In recent years, silver nanoparticles have emerged as potent antimicrobial agents capable of combating extensively drug-resistant pathogenic bacteria that pose serious health risks. The primary aim of our research was to explore the green synthesis of AgNPs using Candida glabrata as an antibacterial agent. A single clinical isolate of Candida glabrata was re-examined via traditional yeast identification methods. Biosynthesis of AgNPs was accomplished by incubating Candida glabrata cell-free supernatant with silver nitrate. AgNP formation was verified by UV-Vis spectroscopy, and the XRD technique assessed the physical properties of the lyophilized AgNPs. EDX and SEM provided insights into the AgNPs’ composition, shape, and size. The antibacterial efficacy was evaluated against pathogenic bacteria through the Agar Well Diffusion method. The formation of AgNPs was evidenced by a shift in color to dark brown. The formation of AgNPs at an absorbance wavelength of 430 nm revealed a polycrystalline structure with an average crystal size of 21.91 nm. The silver constituted 29.50% of the composition and indicated a spherical shape with sizes ranging from 74.96 to 100.40 nm. Significant antimicrobial activity was obtained against pathogenic bacteria. Hence, the proposed research highlights a single-step, cost-effective, and environmentally friendly AgNP synthesis approach that exhibits considerable antibacterial properties.