Purpose: Hyaluronan, commonly known as hyaluronic acid, has been shown to have anti-inflammatory action, bacteriostatic effect and antioxidant properties, thus making its use as a long-term anti-plaque and anti-gingivitis agent an appealing proposition. The aim of the present study was to evaluate the efficacy of 0.025% hyaluronan-containing mouthwash in comparison with 0.2% chlorhexidine and a water-based mouthwash and also to evaluate its antibacterial efficacy on isolated strains of periodontopathogens. Materials and Methods: Forty-five volunteers in a hospital setting were recruited. A single-blinded, parallel design, randomised controlled trial was carried out and the 4-day plaque re-growth model was used to study the efficacy of the three mouthwashes. Microbiological and clinical evaluation was performed by culturing and using dental indices, respectively. The three mouthwashes used in the present study were commercially available 0.025% sodium hyaluronate, 0.2% chlorhexidine and a water-based rinse (negative control). Effects of the three mouthwashes were tested on the growth of isolated strains of Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa) and Prevotella intermedia (Pi). Results: In vitro, hyaluronan had a distinct effect on the growth of Aa and Pi with no effect on the growth of Pg. In vivo, the differences between the individual rinse solutions and the water-based solution showed significantly less plaque regrowth with respect to both chlorhexidine (P = 0.033) and hyaluronan (P = 0.045) when compared to the negative control. The difference between chlorhexidine and hyaluronan was not statistically significant (P = 0.69). Conclusions: Hyaluronan (0.025%)-containing mouthwash was comparable to chlorhexidine (0.2%) in inhibiting plaque growth in vivo, and it significantly reduced the growth of Aa and Pi in vitro. Keywords: anti-inflammatory, antioxidant, anti-plaque, hyaluronan, mouthwash