The role of bactibilia as a risk factor for an unfavorable outcome during biliary disease or surgical procedures remains obscure. Our aim was to identify possible risk factors for bactibilia and their possible relations to complications after elective cholecystectomy. As secondary aims, composition and antibiotic resistance patterns were studied. Bile and gallbladder mucosa samples from 358 elective cholecystectomies were collected prospectively between June 2009 and June 2012. Ordinary microbiologic cultures and antibiograms were performed. All pre-operative factors associated with bactibilia were studied by stepwise logistic regression multivariable analysis. The bacteria isolated most frequently from 103 positive cultures were Escherichia coli (21.3%), Enterococcus spp. (14.7%), and Enterobacter spp. (14.7%) with a global amoxicillin-clavulanic acid resistance rate of 53.7%. Age >65 y, male gender, previous instrumentation or disease of the biliary tract, and high American Society of Anesthesiologists score were independent risk factors. No correlation was found between bactibilia and surgical complications. Although the influence of bactibilia in developing surgical complications is limited, its composition and the high rate of resistance can be influential enough to modify antibiotic treatment in biliary tract infections, especially in high-risk patients.