Background: Patients with periampullary tumors frequently undergo endoscopic biliary investigations and biliary drainage (BD) prior to surgery. Recent literature shows a shift of the biliary microbiome toward more resistant bacteria in patients having BD. This study aimed to evaluate the local microbiome and changes induced by BD and related antibiotic exposure and to consider the choice of antibiotic for peri-operative prophylaxis. Methods: A single-center retrospective cohort study included patients operated on for periampullary tumors between January 2013 and November 2017. All patients had intra-operative bile samples taken for culture and peri-operative antibiotic use as well as documentation of complications according to the Dindo-Clavien classification. Results: A total of 37 patients were included. All received pre-operative endoscopy, and 29 (78%) had BD preceded by administration of ceftriaxone or metronidazole. Intra-operative antibiotic prophylaxis consisted of cefuroxime (92%) or ceftriaxone (13%) combined with metronidazole (100%). Bacterial contamination of bile samples was more common in the BD group than in the no biliary drainage (NBD) group (93% vs 38%; p < 0.01). A shift was observed from bile containing mainly Escherichia coli and Streptococcus spp. toward Enterococcus faecalis (0 in the NBD group versus 44.8% in the BD group; p < 0.01), Enterococcus faecium (0 versus 23%; p = 0.3), and Candida albicans (0 versus 34.5%; p = 0.08). Post-operative antibiotic modifications were common. No difference was found regarding Dindo-Clavien complications, post-operative stay, or antibiotic use in the two groups, although one patient in the NBD group who had pre-operative biliary endoscopy with antibiotic prophylaxis developed a fatal septic clot caused by Escherichia coli resistant to cefuroxime. Conclusions: We observed a significant change toward colonization by enterococci and fungi in the microbiome of patients who had pre-operative biliary investigations or drainage with antibiotic prophylaxis. These findings indicate that bile samples should be obtained systematically during surgery for periampullary tumors to guide any post-operative antibiotic therapy and peri-operative antibiotic prophylaxis and might need adaptation to target the modified microbiome.