The aim of this in vitro study was to investigate the effect of different toothpaste ingredients on biofilm volume and vitality in an established non-contact biofilm removal model. A multi-species biofilm comprising Porphyromonas gingivalis, Streptococcus sanguinis, and Fusobacterium nucleatum was grown on protein-coated titanium disks. Six disks per group were exposed to 4 seconds non-contact brushing using a sonic toothbrush. Four groups assessed slurries containing different ingredients, i.e., dexpanthenol (DP), peppermint oil (PO), cocamidopropyl betaine (CB), and sodium hydroxide (NaOH), one positive control group with the slurry of a toothpaste (POS), and a negative control group with physiological saline (NEG). Biofilm volume and vitality were measured using live-dead staining and confocal laser scanning microscopy. Statistical analysis comprised descriptive statistics and inter-group differences. In the test groups, lowest vitality and volume were found for CB (50.2 ± 11.9%) and PO (3.6 × 105 ± 1.8 × 105 µm3), respectively. Significant differences regarding biofilm vitality were found comparing CB and PO (p = 0.033), CB and NEG (p = 0.014), NaOH and NEG (p = 0.033), and POS and NEG (p = 0.037). However, no significant inter-group differences for biofilm volume were observed. These findings suggest that CB as a toothpaste ingredient had a considerable impact on biofilm vitality even in a non-contact brushing setting, while no considerable impact on biofilm volume was found.