Ultrasound guidance for histological diagnosis is in real-time, convenient, and economical. The aim of this study was to determine whether transvaginal ultrasound (TVUS)- and transrectal ultrasound (TRUS)-guided aspiration biopsy allows detection of a malignant pathology of pelvic/pelvic masses. Data of 40 patients with pelvic and pelvic mass lesions by computed tomography or magnetic resonance imaging underwent TVUS- or TRUS-guided biopsy. Tissue samples obtained were assessed on suitability for histopathologic evaluation. The post-biopsy complication was monitored. All the specimens in the pelvic floor, vaginal stump, vaginal fornix, cervix, and posterior wall of the anal canal were adequate for histologic diagnosis. There were no post-biopsy complications. Transvaginal/transrectal ultrasound-guided aspiration biopsy is safe and simple. It can be used for the diagnosis and differential diagnosis of pelvic and pelvic floor lesions in women. Prospective studies are needed to test diagnostic performance across clinical scenarios.