Novel strategies to combat dental biofilms aim at reducing biofilm stability with the ultimate goal of facilitating mechanical cleaning. To test the stability of dental biofilms, they need to be subjected to a defined mechanical stress. Here, we employed an oral care device (Airfloss) that emits microbursts of compressed air and water to apply a defined mechanical shear to 14-day-old dental plaque in 20 healthy participants with no signs of oral diseases (clinical trial no. NCT05082103). Exclusion criteria included pregnant or nursing women, users of oral prostheses, retainers or orthodontic appliances, and recent antimicrobial or anti-inflammatory therapy. Plaque accumulation, before and after treatment, was assessed using fluorescence images of disclosed dental plaque on the central incisor, first premolar, and first molar in the third quadrant (120 images). For each tooth, the pre- and post-treatment plaque percentage index (PPI) and Turesky modification of the Quigley-Hein plaque index (TM-QHPI) were recorded. The mean TM-QHPI significantly decreased after treatment (p = 0.03; one-sample sign test), but no significant difference between the mean pre- and post-treatment PPI was observed (p = 0.09; one-sample t-test). These data are of value for researchers that seek to apply a defined mechanical shear to remove and/or disrupt dental biofilms. Dataset: The dataset is submitted as a supplement Dataset License: CC-BY-NC.