To assess in a cross-sectional clinical study the effect of antibiotics on the diversity, structure and metabolic pathways of bacterial communities in various oral environments in patients with acute primary infections. Samples of saliva (SA), supragingival biofilm (SB) and from the pulp cavity (PC) were collected from teeth with acute primary infections and then grouped according to previous use of antibiotics (NoAtb=no antibiotics [n=6]; Atb=antibiotics [n=6]). DNA sequencing was conducted using MiSeq (Illumina, San Diego, CA, USA). The V1-V3 hyper-variable region of the 16S rRNA gene was amplified. A custom Mothur pipeline was used for 16S rRNA processing. Subsequent analyses of the sequence dataset were performed in R (using vegan, phyloseq and ggplot2 packages) or QIIME. Twelve patients aged from 22 to 56years were recruited. Participants in the Atb group had taken the beta-lactamics amoxicillin (5/6) or cephalexin (1/6) for 2-3days. A total of 332 bacterial taxa (OTUs) were identified, belonging to 120 genera, 60 families and nine phyla. Firmicutes (41%) and Bacteroidetes (38%) were the most abundant phyla in all samples. Taxa clustered significantly by oral site (PCoA analysis; P<0.05, ANOSIM). Use of antibiotics had little effect on this clustering. However, SA, SB and PC had different degrees of richness, diversity and evenness. The greatest diversity was observed in SB samples and the least diversity was observed in PC samples. Metabolic prediction identified 163 pathways and previous use of antibiotics had a major effect on the estimated functional clustering in SA and PC samples. The ecological niche had a strong influence on the bacterial content of samples from various oral sites. Previous exposure to antibiotics may exert an effect on the phylogenetic composition of SA. Metabolic pathways appear to be modulated by antimicrobial agents in SA and PC samples. The dynamics of host/microbial interactions in the apical region and the functional ecology of the infected pulp cavity should be revisited.