Bile sampling without the risk of contamination by pancreatic and duodenal secretions and avoiding unpredictable influences of general anesthesia during biliary surgery on biliary analytics are feasible with percutaneous puncture of the gallbladder. In 207 patients with gallstones, gallbladder puncture was performed under local anesthesia with a 22-gauge spinal needle under continuous real-time ultrasound guidance. Bile samples were investigated for biliary lipids and nucleation time. Complete aspiration of gallbladder bile could be achieved in all patients without complications such as bleeding, bile leak, or inflammation. Of these patients, 11.6% reported mild abdominal problems, 3.4% required analgetics, and in 1.0% biliary colics were observed. Elective cholecystectomy was performed in 1 patient. Of the bile samples, 10.1% were contaminated with bactobilia. Biliary lipids, cholesterol saturation index (CSI), total lipid concentration (TLC), and bacteriological contamination were independent of gallstone number, whereas patients with solitary gallbladder stones exhibited a significantly longer nucleation time (NT) in comparison with those with multiple stones. In patients with gallstones, fine-needle puncture of the gallbladder represents an important diagnostic procedure and can be performed within minutes without major side effects if performed by an experienced sonographer.