Periodontitis is an inflammatory infectious disease that destroys the tooth-supporting tissues. It is caused by multi-species subgingival biofilms that colonize the tooth surface. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia (i.e. "red complex" bacteria) are characteristic subgingival biofilm species. The Triggering Receptor Expressed on Myeloid cells 1 (TREM-1) is a cell surface receptor of the immunoglobulin super-family, with a role in the amplification of pro-inflammatory cytokine production during infection. This study aimed to investigate TREM-1 mRNA expression in gingival tissues from patients with chronic periodontitis, generalised aggressive periodontitis and healthy subjects, and its correlation with the levels of periodontal pathogens in the tissue. A further aim was to investigate the regulation of TREM-1 in human monocytic cells (MM6) challenged with an in vitro subgingival biofilm model. Gingival tissue TREM-1 expression was increased in both chronic and aggressive periodontitis, compared to health, and correlated with the levels of the "red complex" species in the tissue. No significant differences were detected between the two forms of periodontitis. Biofilm-challenged MM6 cells exhibited higher TREM-1 expression and secretion compared to controls, irrespective of the presence of the "red complex". Engagement or inhibition of TREM-1 affected the capacity of the biofilms to stimulate Interleukin (IL)-1β, but not IL-8, secretion by the cells. In conclusion, this study reveals that TREM-1 tissue expression is enhanced in periodontal disease, and correlates with the level of periodontal pathogens. It also provides a mechanistic insight into the regulation of TREM-1 expression and the associated IL-1β production, in biofilm-challenged monocytes.