The present work focusses on development of a safe, inexpensive, and more accessible source for biosynthesis of silver nanoparticles. Four different in-house probiotic isolates, i.e., Lactobacillus pentosus S6, Lactobacillus plantarum F22, Lactobacillus crustorum F11, and Lactobacillus paraplantarum KM1 isolated from different food sources, were used in the current study to check their ability to synthesize silver nanoparticles. All the probiotic-synthesized silver nanoparticles show maximum surface plasmon resonance (SPR) at a peak of 450 nm, which confirms the formation of silver nanoparticles. Scanning electron microscopy (SEM) analysis identified the shape and distribution of silver nanoparticles. Transmission electron microscopy (TEM) revealed the average size of synthesized nanoparticles in the range of 10–50 nm, with the smallest size of 5 nm for silver nanoparticles synthesized by L. crustorum F11. Further, Fourier-transform infrared spectroscopy (FTIR) detected the presence of different functional groups responsible for reduction of silver ion to form silver nanoparticles. The antimicrobial activity of these AgNPs was also found to be effective against different bacterial and fungal pathogens, viz., antibiotic-resistant Staphylococcus aureus , Bacillus cereus , Listeria monocytogenes , Pythium aphanidermatum , Fusarium oxysporum , and Phytopthora parasitica. However, L. crustorum F11–synthesized AgNP showed maximum inhibition against all the bacterial and fungal pathogens, with highest against S. aureus (20 ± 0.61 mm) and F. oxysporum (23 ± 0.37). Findings from this study provide a durable and eco-friendly method for the biosynthesis of silver nanoparticles, having strong antimicrobial activity against different multidrug-resistant microorganisms. Graphical abstract