Oral spirochetes are anaerobes known as one of causative agents for periodontal diseases. In this study, we investigated the possibility of utilizing fluorescent fatty acids for labeling oral spirochetes. Bacterial labeling was standardized with three different lengths of fluorescent fatty acids: 5-octadecanoylaminfluorescein (OAF), 5-dodecanoylamin-fluorescein (DAF), and 5-hexadecanoylaminfluorescein (HAF). Among these fatty acids, OAF showed the best labeling activity. Treponema denticola ATCC 35405 was totally saturated to the maximum when incubated with OAF 1μM/mℓ for 1 hour. Treponema vincentii LA-1 also increased in fluorescence in proportion to incubation time length and the concentration. In conclusion, these findings showed the possibility that the fluorescent fatty acid can be used for labeling oral spirochetes.