Background: With the growing demand for sustainable dental practices, chitosan has evolved as an eco-friendly disinfectant for polyether impressions. Objectives: The present study investigated the disinfectant efficacy of chitosan on the dimensional accuracy of polyether (PE) impression material. Methods: A total of 63 polyether impressions (Impregum^TM, Penta^TM, 3M ESPE, Boca Raton, FL, USA) were prepared from the master 3D-printed model, each consisting of a single unit abutment facing two units’ abutments. Three groups of these subjects were used, while each group comprised 21 impressions, respectively. Group 1 is used as a control group and is not subjected to chemical disinfection. Group 2 is spraying group, in which the PE impressions were sprayed with 0.5% chitosan with a high molecular weight. Group 3 is the immersion group, in which the impression was immersed for 15 min in 0.5% chitosan with a high molecular weight. The data collected were analyzed using SPSS 28.0 and the difference in dimensional accuracy between the groups was measured using a one-way ANOVA. Results: Both the intra-and inter-abutment measurements (MD and OG) showed no statistically significant differences in the dimensional changes between the control and the study groups, while the cross-arch distance showed a statistically significant difference in the dimensional change between control and immersion group, p = 0.000. Conclusions: While chitosan disinfection induced slight dimensional changes in polyether impressions, these alterations remained within clinically acceptable limits. The spray application method appeared to be preferable to immersion, as it resulted in less pronounced dimensional changes.