Purpose: To verify whether the parts of dental implants can be contaminated by opportunistic pathogens. Materials and Methods: In this cross-sectional study, 38 individuals (52 implants) were investigated. Samples for microbiologic analysis (for a total of 180 sites) were obtained from each individual, from three types of sites in the following order: (1) the peri-implant sulcus of each implant, (2) the gingival sulcus of adjacent teeth, and (3) inside the implant-abutment connection and the abutment of each implant. Swabs from the oral mucosa (cheeks, tongue, and pharynx) were also collected. Quantitative real-time polymerase chain reaction was carried out for total bacterial counts of Enterococcus faecalis and Pseudomonas aeruginosa. Two-way analysis of variance (sites, species) and Holm-Sidak tests were used for statistical analyses. Results: No opportunistic bacteria were found in the gingival sulcus specimens (38 sites). E faecalis was detected in the peri-implant sulcus (3 of 52 sites) and the inner connection/abutment portion (3 of 52 sites). P aeruginosa was identified only in the oral mucosa swabs (1 of 38 sites) and represented the highest bacterial number (3.5 × 106). Statistically significant differences were only found between species and in the peri-implant sulcus. Conclusion: Within the limitations of this study, significant differences in the presence and levels of nosocomial bacteria were detected in the peri-implant environment of diseased implants. From a clinical point of view, data from this study might suggest that, in patients affected by peri-implantitis, prostheses should be removed and the implant-abutment connection disinfected routinely, along with implant surface decontamination. Keywords: implant connection, microbiologic analysis, opportunistic pathogens, peri-implant disease, peri-implantitis