The aim of this study was to examine trends in toothbrushing and inequalities in toothbrushing among girls and boys in Scotland between 1998 and 2006. A secondary aim was to investigate the association between the health promoting school (HPS) initiative and toothbrushing. Data from the Health Behaviour in School-aged Children 1998, 2002 and 2006 surveys were analysed using multilevel logistic regression for boys and girls aged 11, 13 and 15 years. Girls' twice-a-day toothbrushing increased with age while that of boys' remained stable. Toothbrushing increased significantly between 1998 and 2006 for all but 15-year-old girls. Family structure was significantly associated with toothbrushing for 11-year-old boys and 13-year-old boys and girls. Socio-economic inequalities in toothbrushing were significant for both boys and girls at all ages. Largest inequalities were seen among 13-year-old girls and 15-year-old boys. Inequalities persisted over time for all but 15-year-old boys who saw a significant reduction between 1998 and 2006. The HPS initiative in schools in deprived areas was associated with increased odds of twice-a day toothbrushing among 11-year-old boys and 15-year-old girls.