The aim of this study was to compare acute action by killing or disrupting oral biofilms through the use of antimicrobial toothpastes and mouthrinses in vitro and to investigate substantive action by absorption of antimicrobials in a biofilm. Biofilms from freshly collected human saliva were grown in 96-well microtitre plates. After removal of saliva, the wells of the microtitre plates were washed with sterile water (control), or exposed to a dilution series of mouthrinses (Corsodyl, Listerine, Meridol, Crest Pro Health) or to toothpaste slurries (Prodent Coolmint, Colgate Total, Zendium Classic, Crest Pro Health, Oral B Pro Expert, Crest Cavity Protection). Acute action was concluded from reduced continued (16 h) growth of treated biofilms with respect to the control. Substantive action was studied by exposing dead biofilms to mouthrinses or to toothpaste slurry. Substantive action through the absorption and subsequent release of antimicrobials from biofilm was concluded from reduced growth on top of the treated biofilms. All formulations showed acute action at the highest concentrations studied. Further dilution yielded loss of efficacy, or even stimulation of biofilm growth. Antimicrobial absorption in and the release of effective concentrations of antimicrobials from dead biofilms, was demonstrated for three selected antimicrobial products, indicating that antimicrobials remain bio-available for substantive action on new biofilms.