Objectives: Toothbrushing, though aimed at biofilm removal, also affects the lubricative function of adsorbed salivary conditioning films (SCFs). Different modes of brushing (manual, powered, rotary-oscillatory or sonically driven) influence the SCF in different ways. Our objectives were to compare boundary lubrication of SCFs after different modes of brushing and to explain their lubrication on the basis of their roughness, dehydrated layer thickness, and degree of glycosylation. A pilot study was performed to relate in vitro lubrication with mouthfeel in human volunteers. Materials and methods: Coefficient of friction (COF) on 16-h-old SCFs after manual, rotary-oscillatory, and sonically driven brushing was measured using colloidal probe atomic force microscopy (AFM). AFM was also used to assess the roughness of SCFs prior to and after brushing. Dehydrated layer thicknesses and glycosylation of the SCFs were determined using X-ray photoelectron spectroscopy. Mouthfeel after manual and both modes of powered brushing were evaluated employing a split-mouth design. Results: Compared with unbrushed and manually or sonically driven brushed SCFs, powered rotary-oscillatory brushing leads to deglycosylation of the SCF, loss of thickness, and a rougher film. Concurrently, the COF of a powered rotary-oscillatory brushed SCF increased. Volunteers reported a slightly preferred mouthfeel after sonic brushing as compared to powered rotating-oscillating brushing. Conclusion: Deglycosylation and roughness increase the COF on SCFs. Clinical relevance: Powered rotary-oscillatory brushing can deglycosylate a SCF, leading to a rougher film surface as compared with manual and sonic brushing, decreasing the lubricative function of the SCF. This is consistent with clinical mouthfeel evaluation after different modes of brushing.