Understanding deep-time marine biodiversity change under the combined effects of climate and connectivity changes is fundamental for predicting the impacts of modern climate change in semi-enclosed seas. We quantify the Late Miocene–Early Pliocene [11.63 to 3.6 million years (Ma)] taxonomic diversity of the Mediterranean Sea for calcareous nannoplankton, dinocysts, foraminifera, ostracods, corals, molluscs, bryozoans, echinoids, fishes, and marine mammals. During this time, marine biota was affected by global climate cooling and the restriction of the Mediterranean’s connection to the Atlantic Ocean that peaked with the Messinian salinity crisis. Although the net change in species richness from the Tortonian to the Zanclean varies by group, species turnover is greater than 30% in all cases, reflecting a high degree of reorganization of the marine ecosystem after the crisis. The results show a clear perturbation already in the pre-evaporitic Messinian (7.25 to 5.97 Ma), with patterns differing among groups and subbasins.