Objective: In this study, we developed a stationary saliva-derived biofilm model for simultaneous comparison of treatment efficacy of different therapeutic agents (essential oils (EOs), cetylpyridinium chloride (CPC), chlorhexidine (CHX), sodium-(NaF), amine-(AF) and stannous (SF) fluorides).Methods: To test efficacy of the antimicrobial agents under different conditions, biofilms were grown overnight and for 65 hrs, respectively.Overnight grown bacterial communities formed network-like biofilms, while biofilms after 65 hrs of growth were very dense and compact.Therapeutic solutions containing above agents individually or in combination were applied as single treatments for 30 and 60sec, respectively, to overnight grown biofilms and as duplicate 30 sec treatments to mature biofilms.These duplicate treatments were separated by a six hour period of intermittent hourly washes.Confocal laser scanning microscopy and live/dead viability stains were employed to assess treatment efficacy via quantitative evaluation of three-dimensional live/dead cell distribution throughout the biofilm with the analysis software COMSTAT.Results: Against overnight biofilms, EOs were most effective followed by CPC/CHX.The killing efficacy of CPC/NaF1 was less than CPC/CHX but equaled CHX alone, while CPC alone only minimally reduced viability and AF/SF exhibited no significant antimicrobial activity against the biofilms tested.Interestingly, the duplicate treatment of mature biofilms produced very different results: CHX and OEs were most effective and produced similar results, followed by CHX/CPC and AF/SF, while CPC alone did not appear to be very effective under the experimental conditions tested.The antimicrobial activity of CPC/NaF appeared to depend on the overall formulation of the therapeutic agent since efficacies comparable to AF/SF and CPC alone were observed for the different products tested.Conclusion: The biofilm models developed in this study are versatile tools to explore the behavior of different therapeutic formulations under a range of treatment conditions and assess the threedimensional distribution of their killing efficacy.Efficacy of antimicrobials depends on application protocol and biofilm growth phase.