Zinc oxide nanoparticles (ZnONPs) are enormously popular semi-conductor metal oxides with diverse applications in every field of science. Many physical and chemical methods applied for the synthesis of ZnONPs are being rejected due to their environmental hazards. Therefore, ZnONPs synthesized from plant extracts are steered as eco-friendly showing more biocompatibility and biodegradability. Additionally, various synthesis conditions such as the type of precursor salt also play a role in influencing the physicochemical and biological properties of ZnONPs. In this study, green synthesis of ZnONPs from Acacia nilotica was carried out using zinc acetate (ZA-AN-ZNPs), zinc nitrate (ZN-AN-ZNPs), and zinc sulfate (ZS-AN-ZNPs) precursor salts. Surprisingly, characterization of ZnONPs using UV-visible spectroscopy, TEM, XRD, and EDX revealed the important role precursor salts played in influencing the size and shape of ZnONPs, i.e., 20-23nm spherical (ZA-AN-ZNPs), 55-59nm triangular (ZN-AN-ZNPs), and 94-97nm nano-flowers (ZS-AN-ZNPs). FTIR analysis showed the involvement of alkaloids, alcohols, carboxylic acid, and phenolic compounds present in Acacia nilotica extract during the synthesis process. Since different precursor salts showed different morphology of ZnONPs, their biological activities were also variable. ZN-AN-ZNPs showed the highest cytotoxicity towards HepG2 cells with the lowest cell viability (28.92 ± 0.99%), highest ROS/RNS production (3425.3 ± 184.58 relative DHR123 fluorescence), and loss of mitochondrial membrane potential (1645.2 ± 32.12 relative fluorescence unit) as well as induced significant caspase-3 gene expression. In addition to this, studying the zone of inhibitions and minimum bactericidal and inhibitory concentrations of ZnONPs showed their exceptional potential as antibacterial agents. At MIC as low as 8µg/mL, ZA-AN-ZNPs and ZN-AN-ZNPs exhibited significant bactericidal activities against human pathogens Klebsiella pneumoniae and Listeria monocytogenes, respectively. Furthermore, alkaline phosphatase, DNA/RNA leakage, and phosphate ion leakage studies revealed that a damage to the bacterial cell membrane and cell wall is involved in mediating the antibacterial effects of ZnONPs.