Here, we present the synthesis of rod-shaped zinc oxide (ZnO) nanoparticles (NPs) through the wet-chemical method and green synthesis method (using Azadirachta indica leaf extract). Physicochemical properties of the ZnO NPs are characterized using different techniques, and their rod shape structure is confirmed via scanning electron microscope (SEM). Biological effects of the rod-shaped ZnO NPs in human lung (A549) cancer cells are evaluated, which include: viability, apoptosis/cell death, and morphological impact. It has been observed that ZnO NPs synthesized via the green method (marked as L2-NPs) have significantly decreased viability of A549 cells, as compared to NPs synthesized via the wet-chemical method (marked as ZnNPs), ascribed to the presence of therapeutic biomolecules (including polyphenols) in leaf extracts as capping agents. It has also been witnessed that the viability of A549 cells can be significantly reduced in a dose-dependent manner with L2-NPs as well as with only extract of leaf of Azadirachta indica, an IC50 value of 130.95 μg/mL is obtained. Treatment of A549 cells via L2-NPs has effectively (i) improved apoptotic induction post-incubation and (ii) arrested cell cycles in different phases, where a high cell cycle arrest percentage of 83.1% is obtained in the G1-phase itself. Thus, rod-shaped ZnO NPs synthesized via Azadirachta indica leaf extracts have proven to be a potential therapeutic candidate for lung cancer treatment.