The involvement of bacterial translocation (BT) in the promotion of carcinogenesis has gained a considerable attention in the last years. At this point however BT has not been studied in the context of pancreatic cystic lesions and their development into pancreatic ductal adenocarcinoma. The aim of the study was to analyze if bacteria are present in pancreatic cyst fluid (PCF) collected from patients with suspected pancreatic cysts . Total DNA was isolated from sixty nine PCF. The occurrence of bacteria in PCF was analyzed using bacterial 16S rRNA gene-specific PCR-based method followed by sequence identification and quantitative PCR assay tuned up to different pathogenic and commensal human bacteria. Forty-seven out of sixty-nine samples (68%) were found positive for harboring bacterial 16S rRNA gene. Follow up sequencing analyses of the PCR products revealed that bacterial species related to Fusobacterium spp., Bacteroides spp., and Bacillus spp. were predominating the PCF samples. The results suggest that specific bacteria can translocate to the pancreas and become detectable in the PCF.