Though spirochetes have been repeatedly found in marine sponges and other invertebrates, little attention has been paid to the specificity of this association. This study demonstrates that different geno- and morphotypes of spirochetes can reside within the same sponge individual and develop in considerable numbers. Specimens of the calcareous sponge Clathrina clathrus collected from the Adriatic Sea off Rovinj (Croatia) were found to harbor spirochete-like bacteria, which were characterized by scanning electron microscopy (SEM), 16S rRNA gene analysis, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Two novel spirochete sequence types related to the Brachyspiraceae could be retrieved. By use of specifically designed CARD-FISH probes, the C. clathrus-associated sequences could be assigned to a linear and a helical spirochete morphotype. Both were located within the sponge mesohyl and resembled the spirochete-like cells identified by SEM. In addition, from a Clathrina sp., most likely C. coriacea, that originated from Indonesian coastal waters, four different spirochete type sequences were recovered. Two of these also affiliated with the Brachyspiraceae, the other two were found associated with the Spirochaetaceae, one with the genera Borrelia and Cristispira.