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Mechanical plaque control methods such as brushing and flossing are highly recommended to remove dental plaque. The aim of this study is to evaluate the efficacy of the sequence of brushing and flossing on reducing interdental plaque and increasing fluoride retention in that area. This randomized controlled crossover trial was conducted on 25 dental students. After prophylaxis, they were asked to discontinue all forms of oral hygiene for 48 hours. The study was performed in two phases with two‐week washout intervals. In one phase, they first brushed, then flossed (sequence 1: brush‐floss group). In the other phase they initially used dental floss then brushed (sequence 2: floss‐brush group). At each phase, dental plaque (using the Rustogi Modified Navy Plaque Index) and fluoride concentrations (using a fluoride ion specific electrode) were measured before and after flossing and brushing, and the dental plaque reduction and fluoride increase were compared between the two groups using the mixed model test. A significance level of 5% was selected. In the floss‐brush group interdental and whole plaque was reduced significantly more than the brush‐floss group (p= 0.001, p= 0.009 respectively). However, marginal plaque did not show any statistically significant difference between the two groups (p= 0.2). Fluoride concentrations in interdental plaque were significantly higher in the floss‐brush group than the other group (p= 0.027). The results showed that flossing followed by brushing is preferred to brushing then flossing in order to reduce interdental plaque and increase fluoride concentration in interdental plaque.