This study aims to evaluate the imaging findings of cone-beam computed tomography (CBCT) in displaying subtle structures of the tympanic segment of the facial nerve canal in human cadaver heads compared with multi-slice computed tomography (MSCT). Between January 2017 and July 2017, images of the tympanic segment of the facial nerve canal acquired from 73 human cadaver ears by both CBCT and MSCT were prospectively studied. Then, images of the lateral and inferior walls of the tympanic segment were scored using standard imaging slices through a four-point rating scale. Subsequently, the detailed findings of these two imaging modalities were recorded and compared, including interruptions of the bony walls, thread-like bony tubes connected with the walls, and separations in the cavity. The Wilcoxon signed-rank test was used to investigate the differences between scores derived by CBCT and MSCT. The mean score in the inferior and lateral walls by CBCT were significantly higher than that by MSCT (P = 0.000-0.005), which ranged from 2.0 (1.5, 2.5) to 3.5 (3.0, 4.0), and from 1.5 (1.0, 2.0) to 3.5 (2.5, 4.0), respectively. The interruption of the walls was most common at the anterior part of the inferior wall (38/73 cases). Furthermore, thread-like bony tubes were evident in 18 ears, which connected with the anterior part of the inferior wall (18/73 cases). Moreover, separation was found in 22 ears in the posterior part (22/73 cases). CBCT can readily demonstrate subtle imaging findings of the tympanic segment of the facial nerve canal.