Purpose: To evaluate the efficacy of brushing and flossing order on control of plaque and gingival inflammation. Materials and Methods: This cross-over clinical trial was conducted on 35 students recruited from the dental school at Hamadan University of Medical Sciences. The subjects were asked to use dental floss and then brush for a 2-week period (FB), followed by brushing and then using dental floss for another 2-week period (BF). For a 7-day washout interval between the two phases, the subjects practiced dental hygiene according to their habitual method. The participants received dental prophylaxis at the beginning and plaque control record (PCR), plaque index (PI) and bleeding point index (BPI) were measured at baseline and the end of both phases. The differences of indices between two phases were analysed with the Wilcoxon signed rank test; to assess the influence of sex and sequence on PCR, PI and BPI, analysis of covariance (ANCOVA) was used after adjustment of initial values. Results: The PCR and PI indices in the FB method were significantly lower than with the BF method (P 0.05). The PI and PCR index differences between the two phases were significant in men but nonsignificant in women. The effect of gender on PI and BPI and the interaction effect of sex and sequence of brushing and flossing on BPI were significant (P 0.05). Conclusions: Flossing followed by brushing provides more statistically significant improvements over brushing followed by flossing with respect to plaque control. Keywords: dental brush, dental floss, dental hygiene, dental plaque, gingival bleeding