In the present study, zinc oxide nanoparticles (ZnO-NPs) were synthesized using neem leaf aqueous extracts and characterized using transmission electron microscopy (TEM), ultraviolet visible spectroscopy (UV-Vis), and dynamic light scattering (DLS). Then compare its efficacy as anticancer and antibacterial agents with chemically synthesized ZnO-NPs and the neem leaf extract used for the green synthesis of ZnO-NPs. The TEM, UV-vis, and particle size confirmed that the developed ZnO-NPs are nanoscale. The chemically and greenly synthesized ZnO-NPs showed their optical absorbance at 328 nm and 380 nm, respectively, and were observed as spherical particles with a size of about 85 nm and 62.5 nm, respectively. HPLC and GC-MS were utilized to identify the bioactive components in the neem leaf aqueous extract employed for the eco-friendly production of ZnO-NPs. The HPLC analysis revealed that the aqueous extract of neem leaf contains 19 phenolic component fractions. The GC-MS analysis revealed the existence of 21 bioactive compounds. The antiproliferative effect of green ZnO-NPs was observed at different concentrations (31.25 µg/mL–1000 µg/mL) on Hct 116 and A 549 cancer cells, with an IC50 value of 111 µg/mL for A 549 and 118 µg/mL for Hct 116. On the other hand, the antibacterial activity against gram-positive and gram-negative bacteria was estimated. The antibacterial result showed that the MIC of green synthesized ZnO-NPs against gram-positive and gram-negative bacteria were 5, and 1 µg/mL. Hence, they could be utilized as effective antibacterial and antiproliferative agents.