Purpose: To study the relationship between the tooth-brushing habits during the preschool ages and caries prevalence at the age of 5 years and to investigate a possible association with the mode of delivery. Methods: The study population consisted of 336 children that were orally examined at 2, 3 and 5 years. All stages of caries were scored on tooth and surface level. Data on tooth brushing behaviour were collected through semi-structured interviews. Results: At 5 years, the attrition rate was 13.1%. The total caries prevalence (cavitated and non-cavitated lesions) was 18.9%. The vast majority of the parents assisted their child with the brushing and 98% used fluoride toothpaste. The relative risk (RR) for caries was significantly (p < 0.05) increased for “tooth brushing less than twice daily” at two (RR 2.1, 95% CI 1.3–3.3, p < 0.01) and 3 years (RR 3.6, 95% CI 2.0–6.7; p < 0.001). Likewise, reporting “major/minor difficulties to perform tooth brushing” at 2 and 3 years was significantly related to caries development at the age of five (RR 1.5, 95% CI 1.0–2.4, p < 0.05 and RR 2.5, 95% CI 1.4–4.3; p < 0.01). We found no significant association between the tooth brushing habits and the mode of delivery. Conclusions: Less than twice daily tooth-brushing and difficulties to perform the procedure during the first preschool years were significant determinants of caries prevalence at the age of 5 years. Health professionals should, therefore, give special attention and assist parents to improve and optimize their tooth brushing behaviour during the preschool years.