Summary Background Knowledge of the bacterial spectrum for acute cholangitis is essential for adequate empiric antibiotic treatment. Aim To analyse the relation of proton pump inhibitors (PPI) with biliary pathogens in patients with acute cholangitis. Methods This retrospective study identified 278 patients with 318 acute cholangitis episodes using an endoscopic database. The relationship between PPI and microbiological outcomes was assessed by logistic and poisson regression analysis for binary and count data. Results In total, 882 pathogens were isolated, of which, 120 cholangitis episodes occurred with PPI; 198 cholangitis episodes without PPI. Multivariate poisson regression analysis showed that PPI use resulted in a 23% increase in the number of biliary pathogens [3.14 vs. 2.55 (Δ = 0.59), P