Plumeria obtusa (L) is a traditional herb with medicinal properties used worldwide. Plant extracts are the potential precursors for the synthesis of nanomaterials. In this study, we used Plumeria obtusa (L) methanolic leaves extracts to biosynthesize zinc oxide nanoparticles. GC-MS, HPLC, UV- visible spectrophotometer, FTIR, SEM-EDX were used to analyze the phytochemical variations, physicochemical alterations, antibacterial properties. GC–MS and HPLC revealed the presence of strong antioxidant substances like octadecadienoic acid, hexadecanoic acid and benzofuran compounds with the polyphenolic compounds like coumaroylquinic acid, quercetin rutinoside, kaempferol rutinoside and chlorogenic acid. UV– visible spectra of ZnO NPs resulted with absorption at 383 nm. FTIR spectra showed the nearness of extending vibrations of O– H, C=Cl, N=O, C≡C groups at the side C–H and N–H including biomolecules extricates deformation and stabilization of ZnO nanoparticles. SEM images showed irregular and mostly hexagonal shaped morphologies, EDX analysis determined its elemental compositions. ZnO NPs displayed antibacterial activities on Bacillus, Klebsiella, E.coli, Staphylococcus and Enterobacter. In the present study, callus culture was raised for Plumeria obtusa. (L). The results illustrated that leaves obtained forms in vitro raised bulging of tissues with maximum 1 mg/L 2, 4 D and 1 mg/L IAA