The application of nanotechnology in medicine has recently been a breakthrough in therapeutic drugs formulation. This paper presents the structural and optical characterization of a new green nano-formulation (ZnO-Aloe vera) with considerable antibacterial activity against pathogenic bacteria. Its particle structure, size and morphology were characterized by XRD, TEM and SEM. And optical absorption spectra and photoluminescence were measured synchronously. Their antibacterial activity against Escherichia coli and Staphylococcus aureus was also investigated using thermokinetic profiling and agar well diffusion method. The nano-formulation is spherical shape and hexagonal with a particle size ranging from 25 to 65 nm as well as an increased crystallite size of 49 nm. For antibacterial activity, the maximum inhibition zones of ZnO and ZnO+A. vera are 18.33 and 26.45 mm for E. coli, 22.11 and 28.12 mm for S. aureus (p<0.05). Considering Pmax, Qt and k, ZnO+A. vera nano-formulation has a significant (p < 0.05) antibacterial effect against S. aureus almost at all concentration and against E. coli at 15 and 25mg/L. ZnO+A. vera nano-formulation is much more toxic against S. aureus than E. coli, with an IC50 of 13.12 mg/L and 21.31 mg/L, respectively. The overall results reveal that the ZnO-A. vera nano-formulation has good surface energy, crystallinity, transmission, and enriched antibacterial activities. Their antibacterial properties are possibly relevant to particle size, microstructural ionization, the crystal formation and the Gram property of pathogens. This ZnO-A. vera nano-formulation could be utilized effectively as a spectral and significant antibacterial agent for pathogens in future medical and environmental concerns.