In this paper, we report our experience of transvaginal ultrasound (TVUS)-guided core biopsies involving 303 patients referred to the gynaecological ultrasound unit of our national comprehensive cancer centre. Adequate histologic specimens were obtained in 299 patients (98.7%). The most common sites of biopsy sampling were the adnexa (29.7%), the vaginal stump or wall (13.5%), the uterus (11.6%) and the peritoneum (10.2%). Malignancy was confirmed in two-thirds of patients (201/303) and a primary malignancy was diagnosed in 111 of the 201 histologically verified malignant cases (55.2%). Interestingly, 23.9% (48/201) of malignant tumours were proven to have a non-gynaecological origin. Among them, gastrointestinal tumours occurred the most frequently (31/48 patients). Three abscesses were discovered following the biopsy procedure, resulting in a complication rate of 1%. In 94 (31%) patients, subsequent surgery allowed the comparison of the ultrasound-guided and surgically obtained histologic results. We found inaccuracy in 12 cases (12.8%), which is discussed in this paper in detail. Sensitivity, specificity, PPV and NPV to diagnose malignancy was 94.8%, 94.1%, 98.7% and 80.0%, respectively. This is the largest study reported to date about the efficacy and safety of TVUS-guided core biopsy in evaluating pelvic lesions giving rise to a suspicion of gynaecological cancer.