We present here a new passive filtration-based culture device combined to a rapid identification with a new electron microscope (Hitachi TM4000) used for the detection and culture of Treponema species from the human oral cavity. Of the 44 oral samples herein cultivated, 14 were found positive for Treponema using electron microscopy and were also culture-positive (31.8%). All were subcultured on agar plates; based on genome sequencing and analyses, ten were species of T. pectinovorum and five of T. denticola The 30 negative samples for Treponema remained culture-negative. In addition, the 14 Treponema species ordered from the DSMZ collection, were also cultured using this dispositive in the T-Raoult culture medium optimized here. Finally, MALDI-TOF MS was used and 30 novel spectra were added to the MALDI-TOF MS database.We successfully developed a new and effective method for treponemal detection, culture and identification.