Background Natural food additives are widely applied to processed foods, but their maximum usage levels and acceptable daily intake have not been established in most cases. Moreover, relatively little information is available about the safety aspects of natural food additives compared with synthetic ones. Objective We evaluated the cytotoxicity of five natural food additives frequently used in South Korea, such as smoke flavors, erythritol, inositol, shellac, and gamma-oryzanol, at practical usage levels to provide basic information about their potential toxicity and for further in vivo studies. Results The cytotoxicity was assessed for cell viability, membrane damage, and reactive oxygen species generation in cultured intestinal cells and intestinal barrier models. The highest concentrations for cytotoxicity evaluation were set based on their maximum usage and daily intake levels from the Item Manufacturing Report and the Korea Health and Nutrition Examination Survey, respectively. The results showed that the natural additives tested did not exhibit cytotoxicity at their maximum usage and daily intake levels; however, the cytotoxicity of smoke flavors could be affected by manufacturing forms. Additionally, the potential antioxidant activity of erythritol, inositol, and gamma-oryzanol was found. Conclusion The natural food additives tested did not exhibit cytotoxicity at their maximum usage levels. However, the cytotoxicity of smoke flavors was affected by manufacturing forms, suggesting the importance of manufacturing process for the safety of natural food additives.