Gallbladder stones are a very frequently occurring condition. Despite bile bactericidal activity, many bacteria have been detected inside the gallbladder, and gallstones facilitate their presence. Between 3% and 5% of the patients with Salmonella spp. infection develop the carrier stage, with the bacteria persisting inside the gallbladder, shedding bacteria in their feces without signs of infection. The aim of this study was to isolate bacteria from Colombian patients with gallstones, using standard culturing methods, and to identify Salmonella spp. carriers by molecular techniques.A total of 149 patients (120 female and 29 male) diagnosed with gallstones who underwent cholecystectomy and who did not have symptoms of acute inflammation were included. Gallbladder tissue and bile were cultured and used for DNA extraction and Salmonella spp. hilA gene detection.Of the 149 patients 28 (19%) had positive cultures. Twenty-one (75%) patients with positive cultures were from Medellin's metropolitan area. In this geographical location, the most frequent isolations were Pseudomonas spp. (38%), Klebsiella spp. (23%), and Proteus spp. (9%) in addition to unique cases of other bacteria. In Apartado, the isolates found were Enterobacter cloacae (50%), Raoultella terrigena (32%), and both Enterobacter cloacae and Raoultella terrigena were isolated in one (18%) male patient. Five (3.3%) of the 149 patients had positive polymerase chain reaction (PCR) results for the hilA gene of Salmonella spp., all of whom were female and residents of the Medellín metropolitan area.The gallbladder microbiota variability found could be related to geographical, ethnic, and environmental conditions.