Anaerobic digestion of food waste is increasingly utilized for bioenergy generation, producing a byproduct known as food waste digestate (FWD), which has potential applications as a fertilizer within the circular economy. However, accumulating numerous pollutants in FWD poses significant challenges to environmental management and human health. The complex nature of these pollutants complicates both targeted and non-targeted chemical analyses, making safety evaluations difficult. To address this, we developed a toxicity evaluation protocol based on comprehensive cellular effects to assess the safety profile of FWD. Our study found that human FHC cells were significantly more sensitive to FWD solutions, with 1.2-, 1.8-, and 1.7-fold greater sensitivity than GES-1, HepG2, and HEK293 cells. We identified oxidative stress levels and the activation of the NF-κB signaling pathway as crucial and sensitive indicators of FWD-induced toxicity. Metabolomics analysis revealed that FWD triggered the activation of the inflammatory mediator regulation of the transient receptor potential channels pathway, indicating a cellular response aimed at mitigating damage through immune repair mechanisms. By comprehensively assessing these cellular and molecular indicators, we can better predict the potential human and environmental risks associated with FWD. This knowledge is essential for establishing safety guidelines and appropriate dilution ratios for FWD reutilization, enhancing environmental management practices within a circular economy framework.