Background Knowledge of the genetic and environmental influences in caries aetiology has relevance for preventive dentistry. Aims This classical twin study compared concordance of mutans streptococci (MS) and lactobacilli (LB) colonization, enamel defects, and caries in a cohort of 4–6‐year‐old mono‐ (MZ) and dizygotic (DZ) twin pairs. Design The twins were examined for prevalence and concordance of enamel opacities and hypoplasia, oral counts of MS and LB, and dental caries. Bacterial counts were assessed using a commercial microbiological kit. Results Thirty‐four MZ and 50 DZ twins (mean gestational age 35.0 ± 2.4 weeks, and birthweight 2.4 ± 0.6 kg) were examined. There were no statistically significant differences between MZ and DZ twins in the prevalence of MS, LB, and enamel hypoplasia. Concordance rates for MS and LB presence and prevalence of enamel defects within MZ and DZ twin pairs were not significantly different. There were more children with caries in DZ compared with MZ twins (18% vs 3%, P = 0.0029), most likely due to increased daily frequency of sugar consumption and less toothbrushing. Conclusions Concordance data from MZ and DZ twins did not demonstrate any statistically significant difference in susceptibility for enamel defects and colonization of MS and LB.