Developing a cost-effective synthesis route for NiO at room temperature with a low calcination temperature (∼200 °C) remains a significant challenge. This study presents a novel, eco-friendly approach for synthesizing zero-dimensional NiO quantum dots (QDs) via a simple coprecipitation method using minimal reagents and energy-efficient processing. The resulting NiO QDs are obtained in powder form, enabling easy handling, storage, and integration into various applications. X-ray photoelectron spectroscopy, photoluminescence, and Raman spectra confirm the presence of interstitial oxygen (O) and nickel vacancies (V), indicative of intrinsic defects. Temperature-dependent conductivity analysis reveals two distinct regions separated by half the Debye temperature (θ), suggesting the formation of small-polaron-like bound Zhang-Rice states. Furthermore, cytotoxicity studies conducted on A549 and HeLa cancer cell lines and L132 normal cells demonstrate selective toxicity toward cancer cells. These findings highlight the potential of defect-engineered NiO QDs for multifunctional applications, including optoelectronics and biomedicine.