Microorganisms can produce various amphiphilic compounds known as biosurfactants, with diverse applications in distinct industries. This study was focused on the biosurfactant production by Limosilactobacillus fermentum HBUAS62516 for the synthesis of silver nanoparticles. The biosurfactant obtained was characterized as glycolipid using FTIR which showed prominent peaks at 2932.3, 1116.3, and 1084.4 cm, indicating major functional groups which was further confirmed using techniques, such as EDS, NMR, and HPLC. Biosurfactant was utilized as the reducing agent for the biosynthesis of silver nanoparticles which was confirmed using UV-Vis spectral measurements that showed maximum absorbance at 421 nm and FTIR revealed peaks at 109 and 665 cm, indicating silver nanoparticle formation. EDS confirmed the presence of silver nanoparticles with a mass percentage of 100.00 ± 4.56%. Dynamic light scattering (DLS) and zeta potential were 87.93 nm and - 21 mV, respectively, indicating stability. The nanoparticles showed significant antibiofilm and antioxidant activity (90.1%). The synergistic antibacterial effect of the biosurfactant and nanoparticles was studied against Staphylococcus aureus and Pseudomonas putida, as well as their antifungal activity against Aspergillus niger, with a MIC value of 12.5 μg/mL. Nanoparticles synthesized using biosurfactants obtained from probiotic bacteria can act as alternative therapeutics to treat infections caused by the biofilm-forming bacteria.