Otolith mass asymmetry can significantly affect the vestibular system functionalities; usually, the X values of mass asymmetry vary between −0.2 and + 0.2 (−0.2 < X < + 0.2). These values can change during a fish life and therefore they are not related to the fish total length. We collected a total of 404 fish specimens from the Gulf of Tunis, including three pelagic species: Sardina pilchardus (Walbaum, 1792) (74 otolith pairs), Trachurus mediterraneus (Steindachner, 1868) (66 otolith pairs), and Chelon auratus (Risso, 1810) (60 otolith pairs) and three benthic species: Gobius niger Linnaeus, 1758 (77 otolith pairs), Mullus barbatus Linnaeus, 1758 (60 otolith pairs), and Trachinus draco Linnaeus, 1758 (67 otolith pairs). The relation between the total length and the otolith mass asymmetry was first calculated and compared, and then was evaluated. The comparison of the otolith mass asymmetry between benthic and pelagic species showed a significant difference (P < 0.05), where the absolute mean value of X does not exceed the critical value (0.2) for all the studied species. No relation has been found between the magnitude of the otolith mass asymmetry and the length in both benthic and pelagic specimens. Environmental factors have an indirect effect on somatic growth and otolith accretion. The significant difference found in this study can be due to the difference between the benthic and pelagic environments.