BACKGROUND: Biofilm coloration can compromise maturation and increase the risk of oral disease in adulthood, though children with colored biofilm do not always demonstrate a poor oral health status. AIM: The microbial compositions of colored and white biofilms in children were compared. DESIGN: Thirty-two dental biofilm samples from 16 children (age < 13 years) were analyzed using 16S rRNA pyrosequencing, then the subjects were divided into severe caries and healthy (caries-free) groups. Correlations between microbiomes and oral health status were also examined. RESULTS: Phylogenetic analysis revealed no distinctly different patterns between colored and white biofilms. In the severe caries group, genus Actinomyces, Cardiobacterium, Kingella, Lautropia, and Veillonella, and family Neisseriaceae were detected, though abundance was significantly different between colored and white biofilm specimens, in contrast to the healthy group. In addition, five colored biofilm samples from the severe caries group contained greater than 15% Actinomyces, which led us to consider that genus to be possibly associated with formation of colored biofilm in children. CONCLUSIONS: Our findings indicate that differences in bacterial composition between colored and white biofilms are higher in individuals with severe caries. Additional research may reveal the significance of colored dental biofilm in children.