Differentiating gallbladder cancer (GBC) from benign inflammatory conditions such as xanthogranulomatous cholecystitis remains challenging because of overlapping imaging features. Misdiagnosis may lead to unnecessarily extensive resection in benign cases or inadequate oncological treatment in malignant cases. Preoperative histological confirmation can improve diagnostic accuracy and support appropriate surgical decision-making. We retrospectively analyzed 28 patients who underwent image-guided percutaneous transhepatic core needle biopsy for radiologically indeterminate GB lesions with hepatic infiltration. The biopsy findings were classified as malignant, suspicious, or benign. Surgical and pathological outcomes were reviewed, and diagnostic performance was assessed. Concordance between serum tumor markers and frozen sections was evaluated. Biopsies yielded adequate tissue in all cases (100%). Histological results were malignant in 3 patients (10.7%), suspicious in 1 (3.6%), and benign in 24 (85.7%). The final pathology confirmed GBC in 4 patients (14.3%) and benign disease in 24 (85.7%). One malignancy was missed on the preoperative biopsy but was detected intraoperatively. The sensitivity, specificity, and overall diagnostic accuracy were 75.0%, 100.0%, and 96.4%, respectively. Major complications were not observed. Tumor marker levels did not differ significantly between the groups. Frozen sections were concordant with the final diagnosis in 8 of 9 cases (88.9%). Image-guided percutaneous GB biopsy is a safe and effective diagnostic tool for differentiating GBC from benign mimickers. When combined with intraoperative frozen section analysis in selected patients, it allows for optimized surgical planning while minimizing the risk of over- or undertreatment.