A huge amount of seabed acoustic reflectivity data has been acquired from the east to the west side of the southern Adriatic Sea (Mediterranean Sea) in the last 18 years by CNR-ISMAR. These data have been used for geological, biological and habitat mapping purposes, but a single and consistent interpretation of them has never been carried out. Here, we aimed at coherently interpreting acoustic data images of the seafloor to produce a benthic habitat map of the southern Adriatic Sea showing the spatial distribution of substrates and biological communities within the basin. The methodology here applied consists of a semi-automated classification of acoustic reflectivity, bathymetry and bathymetric derivatives images through object-based image analysis (OBIA) performed by using the ArcGIS tool RSOBIA (Remote Sensing OBIA). This unsupervised image segmentation was carried out on each cruise dataset separately, then classified and validated through comparison with bottom samples, images, and prior knowledge of the study areas.