In this study Transition Metal (TM) ions (Co, Ni, and Cr) and Cu co-doped Zinc oxide (ZnO) nanoparticles (NPs) had been synthesized by co-precipitation method. XRD spectra of NPs revealed that TM dopants didn't alter the hexagonal wurtzite structure of the ZnO. However, there was a strong correlation found between the ionic radii of the dopants and parameters. The cell parameters 'a' and 'c', bond length, and volume had a direct relationship with the ionic radii of the dopants. Similarly, the TM dopants enhanced the optical absorption of the NPs and the absorption had an inverse relation with electronegativity. Further, In-vitro cytotoxicity revealed Co and Ni-doped NPs are less cytotoxic (40%) on PBMC at lower concentrations (20-80ng), whereas Cr dopants are highly cytotoxic in all concentrations. All three In-vitro assays (MTT, SRB, and DEM) showed similar results for these NPs. A significant level of antibacterial activity was observed (Average of 25mm ZOI) for all NPs against three pathogens Enterobacter aerogenes, Pseudomonas putida, and Bacillus subtilis. Particularly antimicrobial activity against Gram-negative (Enterobacter aerogenes and Pseudomonas putida) was significant than Gram-positive (Bacillus subtillus) bacteria.