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It is well established that biofilm formed on tooth surfaces is the major culprit for caries and periodontal disease development. The critical early event of biofilm formation is the specific interaction of microorganisms with the acquired enamel pellicle. This pellicle is formed from adsorbing proteins from saliva, and perhaps also from gingival crevicular fluid. The purpose of this project was two fold: first, to investigate the extent of serum protein adsorption in a salivary protein environment, and second, to investigate the colonization of salivary pellicles by early microbial colonizers. Hydroxyapatite (HA) was incubated with saliva and serum and adsorbing proteins were identified by LC-ESI-MS/MS. To investigate competition among salivary and serum proteins for adsorption to HA proteins were labeled with specific CyDyes, mixed in various ratios and incubated with HA. The early phase of oral biofilm formation in vivo was studied on teeth exposed to the oral environment. The harvested biofilm samples were analyzed with the Human Oral Microbiome Identification Microarray containing 407 different microbial probes. In the pure saliva- and serum-derived pellicles eighty-two and eighty-four proteins were identified. Concomitant presence of salivary and serum proteins showed that salivary protein adsorbers effectively competed with serum proteins adsorbers for the HA surface. Specifically acidic proline-rich protein, cystatin, statherin and amylase proteins in saliva competed off apolipoprotein, C-reactive protein, peroxiredoxin-1 and albumin. In vivo evidence supported the replacement of serum proteins by salivary proteins. The studies with oral biofilm formed in vivo led to the identification of 92 species with streptococci being the most abundant early colonizers. High frequency detection was furthermore made with Haemophilus parainfluenzae, Gemella haemolysans, Slackia exigua and Rothia species. Eight uncultivated phylotypes were detected. While there is a significant amount of serum protein emanating from the gingival sulcus, their ability to participate in dental pellicle formation is likely reduced in the presence of strong salivary protein adsorbers. Furthermore, the early pellicle colonizers exhibit considerable bacterial diversity and include non-cultivable species. These findings will be helpful in designing target-specific approaches for the prevention of and/or intervention in diseases exhibiting an oral-biofilm-based etiology.