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In the present study, we explore the potential of Fagonia schweinfurthiiHadidi (FS) in-vivo plant material and in-vitro-grown callus as a bio-template for AgNPs synthesis, highlighting its eco-friendly and bio-inspired sustainable approach. The comparative efficacy of natural plant material and its callus culture was assessed for AgNP synthesis by employing both sunlight and microwave irradiation as separate synthesis methods. This yielded four distinct types of AgNPs denoted as CS-AgNPs, PS-AgNPs, CM-AgNPs, and PM-AgNPs. Sunlight-irradiated AgNP synthesis optimized at near to neutral pH (6.6), whereas microwave-assisted synthesis needed a basic pH for synthesis. Optimization of these AgNPs involved varying concentrations of AgNO3, plant aqueous extract (PAE), and callus aqueous extract (CAE) of FS, exposure time to sunlight and microwave radiation, as well as pH adjustments. The particle size of these AgNPs ranged from 5 to 30 nm, having a crystalline nature with a similar fringe width of 0.22 nm, as observed from HR-TEM analysis. These AgNPs showed significant antibacterial properties against the clinical isolates with λmax 420 nm. Importantly, CAE outperformed PAE for the biosynthesis of AgNPs, mainly in the presence of sunlight-producing CS-AgNPs. These CS-AgNPs are rapidly synthesized, well dispersed and stable, showing significant antibacterial activity, and the least environmental phytotoxicity. It promoted the number of secondary roots, seedling DW, and TWC with 100% seed germination. This highlights their sustainable features for exploring industrial eco-friendly nonmanufacturing of AgNPs using callus culture and sunlight irradiation.