Purpose: The rationale of using platelet-rich plasma (PRP) in reconstructive periodontal surgery is to amplify or accelerate the wound healing through the growth factors contained in platelets. On the other hand, bacterial colonisation of membranes may negatively affect the healing process. The aim of this study was to evaluate bacterial contamination of non-bio-resorbable membranes (ePTFE) used for regenerative periodontal therapy of intrabony defects and the clinical attachment level (CAL) gain with or without PRP. Materials and Methods: Seventeen patients were treated with a natural bone mineral (NBM) and guided tissue regeneration (GTR) with an ePTFE membrane (NBM + GTR group; ie, control group), while in another 17 patients PRP was additionally applied (NBM + PRP + GTR group; ie, test group). Furthermore, the retrieved membranes were analysed for the presence of periodontopathogens and data were related to the gain of clinical attachment. In addition, the in vitro sensitivity of selected microbes to PRP was checked by using agar diffusion test. Results: Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were not detected in the PRP group whereas in the controls A. actinomycetemcomitans was detected in five patients (p = 0.022) and P. gingivalis in two cases (difference not statistically significant, p = 0.242). Detection of A. actinomycetemcomitans was not associated with less CAL gain. If the samples were positively tested for Prevotella intermedia/nigrescens and/or P. gingivalis, the CAL gains were lower compared with the negative samples (p = 0.002). PRP did not show any inhibitory effect on bacterial growth in vitro. Conclusion: Within their limits, the present results appear to suggest that the presence of P. intermedia/nigrescens and/or P. gingivalis at the regenerated site may negatively influence the clinical outcomes. However, the potential influence of PRP on bacterial colonisation and the impact on the clinical outcome is still unclear and remains to be elucidated. Keywords: guided tissue regeneration, natural bone mineral, periodontitis, periodontopathogens, platelet-rich plasma