Objective: The initial microbial colonization of dental surfaces has been studied thoroughly by classical ultrastructural and microbiological studies and further analysed by fluorescent methods. Most of these studies, however, do not differentiate between biofilms formed during night and day. The purpose of the study was to perform a quantitative and qualitative analysis of in situ dental biofilms collected during night and day, respectively. We hypothesised that there is a circadian rhythm in the accumulation of bacteria during initial biofilm formation. Methods: Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by eight individuals for 12-h during day and night, respectively. Subsequently, fluorescent in situ hybridization was performed using probes against Streptococcus spp., Actinomyces naeslundii, and all bacteria and analysed by confocal laser scanning microscopy. Quantification of bacteria was done by stereological tools. Results: The study showed a statistically significant difference between the number of bacteria in the two 12-h groups (Wilcoxon signed rank test, p=0.012) with the highest accumulation of bacteria during day time. The qualitative analysis confirmed this difference within all individuals but with large inter-individual variation in the degree of microbial coverage and bacterial composition. Conclusions: The study provides firm evidence that initial biofilm formation decreases during night. Low biofilm accumulation during the night may reflect circadian rhythms in the rate of bacterial cell division and/or circadian rhythms in the rate of salivary flow and associated drops in salivary nutrients. This finding is of great importance when studying quantitative aspects of initial biofilm formation.