BACKGROUND Microbial adhesion and subsequent biofilm formation on endodontic root canal filling materials and sealers lead to survival of microorganisms in treated root canals and subsequently to endodontic treatment failures. The present study focused on initial microbial adhesion to different endodontic filling materials. METHODS The following endodontic biomaterials were tested: AH-Plus, Tubli Seal, gutta-percha, Real Seal SE, EndoREZ, Apexit Plus, GuttaFlow, and dentin. Samples of each material were prepared. Bovine dentin samples were used as a control. The initial adhesions of salivary bacteria as well as the subsequent single species were quantified by determination of colony-forming units (CFUs) and visualized by scanning electron microscopy and confocal microscopy (CLSM): Enterococcus faecalis, Streptococcus mutans, Streptococcus sanguis, Candida albicans, and Prevotella nigrescens. RESULTS Initially adherent microorganisms could be detected and microscopically visualized on each of the materials tested. Considering the values of the CFUs and the covering grade as detected by CLSM, there were significant differences among the materials. Fewer bacteria tended to adhere to Apexit Plus, whereas Real Seal SE and the widely used gutta-percha showed the highest number of adherent bacteria. This tendency was not detected for C. albicans. CONCLUSIONS Endodontic microorganisms have a high affinity to root canal filling materials and sealers, especially to gutta-percha. Because of this high level of bacterial adhesion, subsequent biofilm formation on these materials could be suggested as leading to the persistence of microorganisms in root canals.