Despite advancements in treatment, the squamous cell carcinoma (OSCC) patient survival rate remains stagnant. Conventional therapies have limited effectiveness, necessitating novel agents. Our study aims to synthesize and characterize amorphous calcium phosphate nanoparticles (nACPs), assess their potential cytotoxic effects on premalignant and malignant OSCC cells, and investigate possible mechanisms of action. The morphological features of nACP were investigated by field emission scanning coupled with energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), and particle size distribution (PSD). Then, we examined the effect of nACPs on nanoparticle uptake, cell adhesion, viability, invasion ability, cell cycle, and gene expression. nACP uptake was dose-dependent, induced limited selectivity in cytotoxicity between healthy and malignant cells, and affected cellular adhesion and invasion. Early apoptosis was the predominant type of cell death. The nACP effect on viability was verified by alterations in the genes associated with apoptosis and proliferation. A high concentration of nACP was shown to arrest the cell cycle progression in the G0/G1 phase of both malignant and premalignant cells. This type of nACP justifies the development of a strategy for its potential use as an anti-cancer agent and/or anti-cancer active carrier for various drugs in oral cancer treatments.