Purpose: During biofilm formation, bacterial species do not attach directly onto the enamel surface, but rather onto the salivary pellicle. Salivary pellicle modification with casein and mucin can hinder erosive demineralisation of the enamel, but it should also not promote bacterial adhesion. The aim of our study was to assess whether salivary pellicle modification with casein, or mucin, or a mixture of both proteins (casein and mucin) influence bacterial adhesion, biofilm diversity, metabolism and composition, or enamel demineralisation, after incubation in: (a) a single bacterial model; (b) a five-species biofilm model; or (c) biofilm reformation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Materials and Methods: Enamel specimens were prepared from human molars. Whole-mouth stimulated human saliva was used for pellicle formation. Four pellicle modification groups were established: control (non-modified pellicle); casein – modified with 0.5% casein; mucin – modified with 0.5% mucin; casein and mucin – modified with 0.5% casein and 0.5% mucin. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 6 h or 24 h. Results: After 24 h incubation in the five-species biofilm model, the mucin group presented significantly lower biofilm mass than the control (p = 0.028) and the casein and mucin (p = 0.030) groups. No other differences between the groups were observed in any of the other experimental procedures. Conclusion: Pellicle modification with casein and mucin does not promote in vitro bacterial biofilm formation. Keywords: biofilms, casein, demineralisation, enamel, mucin, salivary pellicle, salivary pellicle modification