The "green synthesis" of nanoparticles (NPs) offers cost-effective and environmentally friendly advantages over chemical synthesis by utilizing biological sources such as bacteria, algae, fungi, or plants. In this context, cyanobacteria and their components are valuable sources to produce various NPs. The present study describes the comparative analysis of physicochemical and antibacterial properties of chemically synthesized (Chem-AgNPs) and cyanobacteria -derived silver NPs (Splat-AgNPs). The physicochemical characterization applying complementary dynamic light scattering and transmission electron microscopy revealed that Splat-AgNPs have an average hydrodynamic radius of ∼ 28.70 nm and spherical morphology, whereas Chem-AgNPs are irregular-shaped with an average radius size of ∼ 53.88 nm. The X-ray diffraction pattern of Splat-AgNPs confirms the formation of face-centered cubic crystalline AgNPs by "green synthesis". Energy-dispersive spectroscopy analysis demonstrated the purity of the Splat-AgNPs. Fourier transform infrared spectroscopy analysis of Splat-AgNPs demonstrated the involvement of some functional groups in the formation of NPs. Additionally, Splat-AgNPs demonstrated high colloidal stability with a zeta-potential value of (-50.0 ± 8.30) mV and a pronounced bactericidal activity against selected Gram-positive ( and ) and Gram-negative ( and ) bacteria compared with Chem-AgNPs. Furthermore, our studies toward understanding the action mechanism of NPs showed that Splat-AgNPs alter the permeability of bacterial membranes and the energy-dependent H-fluxes via FF-ATPase, thus playing a crucial role in bacterial energetics. The insights gained from this study show that -derived synthesis is a low-cost, simple approach to producing stable AgNPs for their energy-metabolism-targeted antibacterial applications in biotechnology and biomedicine.