Guidelines for antibiotic treatment of acute cholecystitis are based on studies using culture techniques for microbial identification. Microbial culture has well described limitations and more comprehensive data on the microbial spectrum may support adjustments of these recommendations. We used next generation sequencing to conduct a thorough microbiological characterization of bile-samples from patients with moderate and severe acute cholecystitis.We prospectively included patients with moderate and severe acute cholecystitis, undergoing percutaneous or perioperative drainage of the gall bladder. Bile samples were analyzed using both culture and deep sequencing of bacterial 16S rRNA and rpoB genes and the fungal ITS2-segment. Clinical details were evaluated by medical record review.Thirty-six patients with moderate and severe acute cholecystitis were included. Bile from 31 (86%) of these contained bacteria (29) and/or fungi (5) as determined by sequencing. Culture identified only 40 (38%) of the 106 microbes identified by sequencing. In none of the 15 polymicrobial samples did culture detect all present microbes. Frequently identified bacteria often missed by culture included oral streptococci, anaerobic bacteria, enterococci and Enterobacteriaceae other than Klebsiella spp. and Escherichia coli.Culture techniques display decreased sensitivity for the microbial diagnostics of acute cholecystitis leaving possible pathogens undetected.