AbstractAim: To compare effects of three cetylpyridinium chloride (CPC) formulations with and without alcohol and Tween80 on physico‐chemical properties of salivary pellicles, bacterial detachment in vitro and bacterial killing in vivo.Material and Methods: Adsorption of CPC to salivary pellicles in vitro was studied using X‐ray photoelectron spectroscopy and water contact angle measurements. Adhesion and detachment of a co‐adhering bacterial pair was determined in vitro using a flow chamber. Killing was evaluated after live/dead staining after acute single use in vivo on 24‐ and 72‐h‐old plaques after 2‐week continuous use.Results: The most pronounced effects on pellicle surface chemistry and hydrophobicity were observed after treatment with the alcohol‐free formulation, while the pellicle thickness was not affected by any of the formulations. All CPC formulations detached up to 33% of the co‐adhering pair from pellicle surfaces. Bacterial aggregate sizes during de novo deposition were enhanced after treatment with the alcohol‐free formulation. Immediate and sustained killing in 24 and 72 h plaques after in vivo, acute single use as well as after 2‐week continuous use were highest for the alcohol‐free formulation.Conclusions: CPC bioavailability in a formulation without alcohol and Tween80 could be demonstrated through measures of pellicle surface properties and bacterial interactions in vitro as well as bacteriocidal actions on oral biofilms in vivo.