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Introduction: Considering the broad spectrum of using nanoparticles in food coatings as a potent antimicrobial agent and their possible cytotoxic effects and accidental consumption of these toxic materials, this study was performed. The present study aimed to investigate the cytotoxicity of chitosan and nano-chitosan in vitro. Material & Methods: This cross-sectional study was conducted in 2019 in the Laboratory of the Department of Food Hygiene and Public Health, School of Veterinary Medicine, Shiraz University, Shiraz, Iran. The XRD and FITR techniques were employed to study the characteristics of these nanoparticles. Moreover, two cell lines, HT-29 and Vero, were used to study the cytotoxic effects of chitosan and nanochitosan by MTT assay, acridine orange, and ethidium bromide staining. One-way ANOVA and independent t-test were used to analyze the collected data with the help of SPSS software (version 19). Results:Based on the obtained findings, the maximum values of XRD at the angle of θ2 were observed at 20°. The highest peak appeared at 1530 cm-1, which was associated with tensile vibration of N-P-N. The spectrum diagram of chitosan due to the tensile vibration of synthetic nanochitosan of N-H bound appeared at 1646 cm-1. The results showed a proportional increase in the cytotoxicity with time and concentration of nanoparticles in the cells. Discussion & Conclusion: Since by increase in time and concentration of nanoparticles, toxicity was observed in cells; therefore, the time and concentration of chitosan nanoparticles are important in causing cytotoxicity. Considering the toxic effects of these nanocomposites on cancer cells, they can be used in cancer treatment, which requires further studies. [ABSTRACT FROM AUTHOR]