Plant-based nanoparticles can be tuned through the frequency of light for efficient synthesis, structural properties, and antibacterial applications. This research assessed the effect of material type (callus and whole-plant extract) and the interaction with a specific range of light wavelength on AgNP synthesis. All types of AgNPs were characterized by their size, shape, associated functional groups, and surface charge. Interestingly, the size of red light and callus-based AgNPs (RC-AgNPs) was smaller (6.32 nm) compared to 14.59 nm for Ultraviolet light and callus-based AgNPs (UV-C-AgNPs). Zeta potential analysis showed that RC-AgNPs had higher stability (-29.2 mV) compared to UV-C-AgNPs (-16.7 mV). Similarly, red light-based AgNPs had higher Oxidation reduction potential in both whole-plant-based and callus-based AgNPs, indicating a more oxidizing nature compared to those synthesized under UV light. This was confirmed by the lower total phenolic and flavonoid content associated with them and their lower antioxidant activity. The higher antibacterial activities and lower minimum inhibitory concentrations of red light-based AgNPs against highly resistant pathogenic bacteria demonstrated the role of red light in enhancing antibacterial activity. These results indicate that AgNPs synthesized in red light and callus extract are more active compared to those synthesized under other wavelengths and/or in whole-plant extracts.