Background: The microbiology of pancreatoduodenectomy is challenging and published guidelines regarding perioperative antimicrobial prophylaxis are variable with poor adherence. Methods: A retrospective analysis of the microbiological results of 294 consecutive patients who underwent pancreatoduodenectomy was performed. Intraoperative specimen culture results were available for 50 patients and their medical records were reviewed to determine the following demographics and factors; age; sex; tumour location, histopathology, grade and stage; neoadjuvant chemotherapy and radiotherapy; preoperative biliary stenting; surgeon; surgery type and antimicrobial prophylaxis coverage. Outcomes assessed included; post-operative infections, mortality (all and 90-day), and intensive care unit and hospital admission durations. Univariate analysis with chi-squared testing was performed. Results: Intraoperative specimen cultures were positive in 48 (96%) patients and polymicrobial in 45 (90%) patients with a predominance of Enterobacteriaceae (38/76%), Enterococcus species (27/54%), and Candida species (25/50%). Isolates were potentially susceptible to the current perioperative antimicrobial prophylaxis regimen of ceftriaxone with or without metronidazole in only six patients. However, only neoadjuvant radiotherapy was associated with statistically significant increased intensive care unit and hospital admission durations. Conclusion: Although this study was probably underpowered to detect any statistically significant associations, perioperative antimicrobial prophylaxis coverage of the operative field microbiological milieu of pancreatoduodenectomy is logical and current guidelines may be inadequate.