The Mediterranean Sea is a renowned biodiversity hotspot influenced by multiple interacting ecological and human forces. A gap analysis on the biology of Mediterranean marine fishes was conducted in 2017, revealing the most studied species and biological characteristics, as well as identifying knowledge gaps and areas of potential future research. Here, we updated this gap analysis five years later by reviewing the literature containing information on the same eight biological characteristics, namely length-weight relationships, growth, maximum age, mortality, spawning, maturity, fecundity and diet, for the 722 fish species of the Mediterranean Sea. The results revealed a considerable knowledge gap as 37% of the species had no information for any of the studied characteristics, while 13% had information on only one characteristic. Out of all the biological characteristics, the smallest knowledge gap was found in the length-weight relationships (studied for 51% of the species, mainly in the eastern Mediterranean), while the least studied characteristic was mortality (studied for 10% of the species). The western and eastern Mediterranean Sea were leading forces in data collection exhibiting the narrowest gaps between current and desired knowledge. The most studied species across the entire region were the highly commercial European hake (Merluccius merluccius), red mullet (Mullus barbatus), European anchovy (Engraulis encrasicolus), European pilchard (Sardina pilchardus), common pandora (Pagellus erythrinus), and annular seabream (Diplodus annularis). The knowledge gap has shrunk by 6% during the last five years, with 40 new species having at least one study on their biology. Moreover, research has slightly shifted towards species that have been traditionally neglected, e.g., sharks, rays and chimaeras (chondrichthyans). It is recommended that research becomes less focused on commercial species and more targeted towards the identified gaps, vulnerable species (e.g., deep-sea species and chondrichthyans) and species that could potentially pose a threat (e.g., non-indigenous species) to the ecosystems of the everchanging Mediterranean Sea.