The Neretva dwarf goby Orsinigobius croaticus (Gobiiformes, Gobionellidae) is an endemic fish native to the freshwaters of the Adriatic Basin in Croatia and Bosnia and Herzegovina. Due to its limited distribution range, specific karst habitat and endangered status, laboratory studies on reproductive biology are scarce. We investigated the sound production and acoustic behaviour of this species during reproductive intersexual laboratory encounters. We performed dissection and micro-computed tomography (μCT) scanning of the pectoral girdle to explore the anatomy of its putative sound producing mechanism. To study interspecific acoustic differences and determine whether acoustic features can discriminate among species, comparative analysis was conducted on sounds produced by closely related soniferous sand gobies. Our results indicate that males of the O. croaticus emit pulsatile sounds composed of a variable number of short (~ 15 ms) consecutive pulses when interacting with females, usually during the pre-spawning phase in the nest, but also during courtship outside the nest. Pulsatile sounds were low-frequency and short pulse trains (~ 140 Hz, < 1000 ms), and spectro-temporal parameters were correlated with physical traits and water temperature. Male visual behaviour rate was higher when co-occurring with sounds and females entered the male’s nest significantly more frequently when sounds were present. Male sound production was accompanied by movements such as head thrust and fin spreading. μCT scans and dissections suggest that O. croaticus shares certain anatomical similarities of the pectoral girdle (osseous elements and arrangement of levator pectoralis muscles) to previously studied sand gobies. Multivariate comparisons, using sounds produced by eight soniferous European sand gobies, effectively distinguished soniferous (and sympatric) species based on acoustic properties. Discrimination success decreased when temperature-dependent features (sound duration and pulse repetition rate) were excluded from analysis. Therefore, we suggest both spectral and temporal features are important for acoustic differentiation of sand gobies.