Chitosan mouthwash has higher antibacterial effects with lower cytotoxicity than many other mouthwashes. For the first time, the present study compared the enamel discoloration of natural teeth by using chitosan, chlorhexidine, and chitosan-chlorhexidine mouthwashes. Method: In this laboratory study, 45 premolar teeth extracted for orthodontic reasons without structural defects, cracks, restorations and observable caries were randomly divided into three groups (Chitosan, Chlorhexidine, and Chitosan-Chlorhexidine) of 15 each. First, prophylaxis pastes and plastic rubbers were used to clean the surfaces of the teeth; then, a spectrophotometer was used to measure the initial color of the buccal surfaces of the teeth. Then the first-, second-, and third-group of teeth were immersed in the 0.5% chitosan solution, 0.2% chlorhexidine solution, and the combined 0.5% chitosan and 0.2% chlorhexidine solution, respectively, for 24 h. Then the color of buccal surfaces of the teeth were again measured by the spectrophotometer, and discoloration (ΔE) was calculated. Statistical analysis was performed by SPSS.26 software, ANOVA analysis of variance and Bonferroni post hoc tests. Results: In the chitosan group, discoloration (3.14±1.86) was significantly higher than those in the chitosan-chlorhexidine group (1.74±0.88) and the chlorhexidine group (1.78±0.58); however, the latter two groups experienced similar discoloration. Conclusion: Chitosan-chlorhexidine mouthwash revealed dental discoloration effects similar to those of chlorhexidine.