Gallbladder mucoceles (GBMs) have become a clinically important cause of extrahepatic biliary disease in dogs, particularly in older patients and certain predisposed breeds. The reported percentage of positive bacterial cultures in cases of GBMs, however, has been variable. This study investigated the use of fluorescence in situ hybridization, a culture-independent technique, for detecting bacteria in GBMs. Fluorescence in situ hybridization was found to be more sensitive than bile culture for detecting bacteria. Of the 25 dogs with GBMs in this study, 68% were also found to have concurrent cholecystitis, a higher percentage than has been reported previously in dogs with GBMs (ie, 17%-40%). The relationship among bacteria, cholecystitis, and the etiology and progression of GBMs remains to be determined.