Background The infection risk of natural orifice transluminal endoscopic surgery (NOTES) is of concern. The aim of this study was to assess the safety of NOTES by investigating the intraperitoneal bacterial load during transgastric and transvaginal procedures with antiseptic or controling perioperative preparation.Methods Forty-five female pigs were randomly assigned to five equal groups: the transgastric (TG) control group (group A), the TG middle volume gastric lavage group (group B), the TG high volume lavage group (group C), the transvaginal (TV) control group (group D) and the TV study group (group E). The study groups received gastric or vaginal lavage and abdominal antimicrobial irrigation, while the control groups received neither. All animals were administered intravenous antibiotics, underwent NOTES peritoneoscopy and transumbilical laparoscopic cholecystectomy under NOTES view with sterile instruments. The viscerotomy was closed by laparoscopic suture. The animals were observed until necropsy was performed 14 days postoperatively. Quantitative bacteriologic cultures were taken from the gastric or vaginal aspirate before and after lavage; peritoneal fluid was collected before and after peritoneal irrigation and at necropsy. Results The surgical procedures were completed for all the pigs and all of them survived. The mean operative time of the TG group and the TV group was (81±27) minutes and (66±12) minutes, respectively. All animals survived for 14 days.At necropsy, significantly more peritoneal infections were noted in group A than in group D (5:9 vs. 0:9; P ＜0.05). Nogross evidence of intra-peritoneal infection was found in groups B, C, D and E. Bacteriological evidence was seen in all pigs in group A, 7 pigs in group B, 6 pigs in group D, and none in groups C and E.Conclusions Without gastric or vaginal lavage and antibiotic peritoneal irrigation, the TG procedure has a higher infection rate than the TV access. After antiseptic preparation, the bacterial load significantly decreased in the TG group,which seems as safe as the sterile TV approach.