Background <p>Chlorhexidine is the most effective plaque and gingivitis control mouthwash. However, it causes some undesirable adverse effects such as tooth staining, alteration in taste, and oral mucosal irritation. To reduce these side effects while maintaining the properties of the mouthwash, we have developed a new mouthwash formulation. Therefore, this study aimed to evaluate the efficacy of 0.001% EDTA containing chlorhexidine mouthwash (CHX + EDTA) in minimizing tooth stain forming, compared to the commercially available 0.12% chlorhexidine mouthwash (CHX). Additionally, dental plaque and gingival inflammation were compared between the two mouthwashes.</p> Methods <p>A double-blind, crossover randomized clinical trial was conducted in 58 healthy participants to compare CHX + EDTA (test group) and CHX (control group). After professional cleaning at baseline, participants were assigned to use a mouthwash twice daily for 14 days, then switched after a 2-week washout. Each participant’s mouth was divided into two sides: one with regular oral hygiene and the other without. The plaque index, gingival index, percentage of sites with bleeding on probing, and staining index were evaluated.</p> Results <p>Among the 58 participants, CHX + EDTA showed significantly less tooth staining than CHX in the extent and intensity of the staining index, on both brushing (1.05 vs. 1.29) and non-brushing sides (1.55 vs. 1.75). Both mouthwashes demonstrated similar efficacy in reducing plaque accumulation and resolving gingival inflammation (<i>p</i> &lt; 0.05).</p> Conclusions <p>The 0.001% EDTA containing chlorhexidine mouthwash significantly minimizes tooth staining and is as efficient as chlorhexidine in reducing dental plaque and gingival inflammation.</p> Trial registration <p>Thai Clinical Trials Registry (TCTR20240830005) registered on 26 June 2024 (TCTR20240830005).</p>