The oral microbiome is ecologically diverse, complex, dynamic, and little understood. We describe the microbiota of four oral habitats in a birth cohort at age 32 and examine differences by sex, oral hygiene, and current smoking status, dental caries, and periodontal health. Oral biofilm samples collected from anterior labial supragingival, posterior lingual supragingival, subgingival, and tongue sites of 841 Dunedin Multidisciplinary Health and Development Study members were analysed using checkerboard DNA-DNA hybridization; focusing on 30 ecologically important bacterial species. The four habitats exhibited distinct microbial profiles that differed by sex. Streptococcus gordonii was more dominant in supragingival and tongue biofilms of males; Porphyromonas gingivalis exhibited higher relative abundance in subgingival biofilm of females. Males had higher scores than females for periodontal pathogens at supragingival sites. The relative abundance of several putative caries and periodontal pathogens differed in smokers and non-smokers. With poor oral hygiene significantly higher proportions of Gram-negative facultative anaerobes were present in subgingival biofilm and there were higher scores for the principal components characterised by putative cariogenic and periodontal pathogens at each site. Distinctive microenvironments shape oral biofilms and systematic differences exist by sex, oral hygiene, and smoking status.