Purpose: Despite being the gold standard antiplaque agent, chlorhexidine (CHX) has many adverse effects that make scientists search for new agents to combat biofilms as effective as CHX. Hyaluronan, also known as hyaluronic acid (HA), is a natural polysaccharide with anti-inflammatory, antioxidant and bacteriostatic properties. The objectives were to evaluate the plaque inhibitory, and anti-inflammatory effects of HA mouthwash compared to CHX and distilled water (DW) in a 4-day non-brushing model together with the participants' preference to the used products. Materials and Methods: Thirty-three systemically and periodontally healthy subjects were included in this randomised, double-blinded, crossover clinical study. Subjects were randomly assigned into three treatment-sequence groups to use three mouthwashes one after another, in three different time periods. After professional prophylaxis at day 1, subjects refrained from all oral hygiene measures and used mouthwashes that were individually allocated to them. On day 5, scoring of plaque index (PI) according to Turetsky modification of Quigley Hein Index system, modified gingival index (MGI) and measurement of gingival crevice fluid (GCF) volume were performed. Treatment satisfaction questionnaire form was given at the end of each experimental period. Results: CHX showed statistically significant reduction in PI followed by HA (p = 0.048). No statistically significant differences were detected between HA and CHX in terms of MGI and GCF volume. For HA, subjects reported significantly better taste, less sensitivity, burning sensation, mouth dryness and numbness perception compared to CHX and DW. Conclusions: CHX revealed the best plaque inhibition closely followed by HA. Early gingival inflammatory changes were found similar for CHX and HA. Furthermore, HA was well accepted with better perceptions than CHX and DW.