The present investigation aimed to explore the potential of different organs and group of cells (calli) of the same plant for the biogenic synthesis of zinc oxide nanoparticles using leaf and aerial part of the plant as organ and leaf derived calli as a group of cells of choice. Zinc nitrate and zinc acetate were used as a precursor for the synthesis of ZnO nanoparticles. The callus was generated on MS basal medium with 2,4-dichlorophenoxy acetic acid (2,4-D) (0.25–3.0 mgl−1) and kinetin (Kn) (0.5–2.0 mgl−1) in all possible combinations as a growth regulator. Maximum callus induction frequency (96.56 ± 0.20%) was reported in cultures having 2,4-D+Kn in 2.0 mgl−1 + 1.0 mgl−1 concentration. The synthesized nanoparticles were preliminarily characterized by UV-visible spectroscopy for their confirmation of synthesis and stability after 10 days. The characterization of synthesized nanoparticles was carried by using XRD, TEM for their crystal shape, size and morphology. XRD analysis suggests crystalline nature with wurtzite hexagonal shape of all synthesized nanoparticles for different plant material used, confirming the potential of Calli as an alternate source of plant material for the synthesis of nanoparticles. TEM analysis suggests the size of nanoparticles, with the smallest nanoparticles (less than 15 nm) were synthesized using aerial parts of the plant. The functional groups present in the plant extract may responsible for the bio-reduction and capping of nanoparticles were characterized using FTIR spectroscopy and results revealed the presence of phytochemicals on the surfaces of synthesized nanoparticles. The antimicrobial activity of nanoparticles was tested against different bacterial strains, i.e., Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumonia, and E. coli and clear zone of inhibition showed their anti-bacterial potential.