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In the current study, parietin was extracted aerobically from lichen and quantified using parietin activity tests. Different parameters were optimized to extract parietin, viz. suspending 1.5 g biomass of lichen in 15 mL methanol and incubating it for 45 min at 37 °C. Parietin-mediated AgNPs were synthesized by 30-minute procurement period with 3 mL of AgNO3 (3 mM) solution. Visual observations and ultraviolet–visible (UV–Vis) spectra demonstrated that the Ag precursor’s treatment changed the color of fresh methanolic extract to greenish-brown. Fourier transfer infrared (FTIR) spectroscopy was used to identify lichen secondary metabolites. The effective synthesis of Xa-AgNPs was further validated by X-ray diffraction patterns and scanning electron microscopy. The free parietin extract and synthesized nanoparticles (Xa-AgNPs) were evaluated for antimicrobial activity against selected bacterial isolates; S. aureus, B. subtilis (gram-positive), E. coli, Pseudomonas sp. (gram-negative) by agar well diffusion method. The results indicated that free parietin extract and synthesized nanoparticles (Xa-AgNPs) have potential antimicrobial activity against selected gram-positive and gram-negative bacterial isolates. The current results exhibited that Xa-AgNPs are potent bactericidal agents and should be considered a promising candidate to combat bacterial infections.