This study aims to evaluate the prevalence of trichomonads in the subgingival biofilm of patients with periodontitis. Secondarily, the trichomonad presence was related to patient characteristics and periodontal clinical parameters, in order to highlight the factor favoring the development of these protozoans. Subgingival biofilm samples were collected from at least two diseased and one healthy site in 50 patients suffering from periodontitis. Trichomonads were identified using phase contrast microscopy. All patient characteristics and periodontal clinical parameter data were then statistically analyzed. From the 50 patients examined, 195 sites were sampled, including 145 diseased ones. Trichomonads were only observed on 16 of the 145 diseased sites (11%) and none in the other 50 healthy sites. Based on these results, 20% (n = 10) of patients were positive for the presence of trichomonads from at least one of the diseased sites collected. Tooth mobility, substantial supra-gingival dental deposits, and severe clinical attachment loss were statistically associated with trichomonad presence. If the subgingival biofilm of male patients over the age of 50 seemed to be more frequently contaminated with trichomonads, this data was not statistically supported. This preliminary study indicates for the first time that in periodontitis-involved patients, trichomonads are observed in the subgingival biofilm collected from diseased sites with severe bone loss, but not from healthy teeth. Further investigations are needed to fully explore the role of this microorganism in the etiology of periodontal disease.