Summary Lipophilic components are known to modulate the process of bioadhesion on the tooth surface. However, the presence of lipid droplets at the acquired pellicle under oral conditions has not been demonstrated, yet. The purpose of the present study was to establish a method for direct visualisation of lipids on the surface of hydrated, pellicle covered tooth samples by environmental scanning electron microscopy (ESEM), and to use this technique for studying the effects of rinsing with edible oils on the acquired pellicle under in vivo conditions. In situ pellicle formation was performed by 3 min exposure of enamel and dentin specimens in the oral cavity of volunteers. Subsequently, the volunteers rinsed in vivo with safflower oil or linseed oil for 30 s, and the specimens were further carried intraorally for periods from 0 min up to several hours. After intraoral exposure the specimens were treated by osmium tetroxide vapour, and were subsequently analysed by ESEM. This technique was capable to directly visualise the presence of lipid droplets at the pellicle's surface under hydrated conditions. ESEM analyses revealed that surface bound nano- and micro-sized lipid droplets were present at the acquired pellicle's surface even several hours after rinsing with edible oils indicating that these droplets had tightly adhered to the pellicle surface. Pellicle modification by edible oil rinsing as demonstrated in the present study might have the potential to be beneficial as an adjunct in dental prophylaxis.