We investigated bacterial colonisation patterns of healthy mucosa (buccal, tongue, palate and floor of mouth) in a cohort of adults in order to determine how smoking, tooth loss, plaque levels and oral hygiene practices impacted on mucosal colonisation. A total of 322 swabs were recovered from 256 participants, of whom 46% were current smokers. We analysed colonization by sequencing the V1-V3 regions of the 16S rRNA gene. Palate and tongue microbiomes generally exhibited greater biodiversity than buccal and floor of mouth. Although , and spp. showed reduced abundance in smokers, buccal mucosa specifically showed a significant increase in spp., whereas tongue and floor of mouth tended towards increased abundance of spp. Unexpectedly, tooth brushing frequency had a greater impact on mucosal community structure than plaque levels. Tooth loss was associated with significant reductions in mucosal biodiversity and had site-specific impacts, with buccal communities showing increased abundance of periodontitis-associated species and , whereas tongue communities exhibited increased abundance of several streptococcal OTUs and reduced abundance of spp. This study highlights the complex relationship between mucosal colonisation and host factors, highlighting the need for careful consideration of these factors in mucosal microbiome studies.