Introduction Secondary biofilm formation by oral bacteria after breakdown/fracture of temporary or permanent restorations imposes a challenge to the outcome of root canal treatment. This study focuses on benzalkonium chloride (BAK) coating on dentin or polystyrene surfaces and its influence on the early adhesion and biofilm formation by oral and root canal bacteria. Methods Microbial adhesion and biofilm growth on surfaces coated with BAK were analyzed qualitatively with a dentin disk model and quantitatively with a mini-flow cell biofilm model. Cell viability and total biovolume were analyzed by the LIVE/DEAD technique. The repelling effect of surfaces coated with BAK was compared with NaOCl. Uncoated surfaces were used as controls. Results Scanning electron microscope images in the dentin disk model revealed that very sparse biofilms were formed on NaOCl- and BAK-coated dentin surfaces. In contrast, biofilms formed on uncoated dentin were clearly visible as numerous irregularly distributed aggregates of rods and cocci. In the mini-flow cell system, confocal laser scanning microscope analysis confirmed that biofilms formed on NaOCl- and BAK-coated surfaces showed significantly less adhesion (2 hours) and biovolume accumulation (24 and 96 hours) compared with the uncoated controls (P < .01). Furthermore, cell viability assessments showed that on uncoated controls the viability measurements were high (>89%) as well as on BAK-coated surfaces (88% viable cells). However, cell viability was significantly reduced on NaOCl-coated surfaces (59% viable cells). Conclusions This study illustrates that surface coating with a surfactant solution containing BAK does not cause cell membrane damage but might interfere with cell mechanisms of adhesion. Investigations into the clinical utility of BAK as an antibiofilm medication are warranted.