Functional groups are sets of species that play a similar role in a food web. We defined functional groups of fish species based on their morphological characteristics, while using expert knowledge for invertebrates. We measured 9 morphological traits of 72 fish species, and carried out multivariate analyses to assign fish species to functional groups. The analysis identified 9 trait-based fish functional groups to which were added 3 expert-based invertebrate functional groups. The habitat (position in the water column) and potential diet of each group were identified from the literature. Using the MEDITS bottom trawl survey data collected at 10 to 800 m depth, we calculated relative change in the 12 fish and invertebrate functional group biomasses for 12 Mediterranean areas over the period 1994 to 2012. Multiple regression trees identified 4 regions with similar changes: (1) the Adriatic and the Ionian Sea; (2) the Tyrrhenian Sea and the Strait of Sicily; (3) the Balearic Islands and other enclosed areas such as the Gulf of Lions and Aegean Sea; and (4) the Ligurian Sea and Sardinia. The biomass of all functional groups increased or remained stable in the first 2 regions, while around half the functional group biomasses decreased in the other 2 regions. These regional differences in functional group biomass changes were mainly associated with regional variations in the time trends of bottom water temperature (37%), bottom water dissolved oxygen (23%) and mean catch levels (9%). This study contributes to the EU Marine Strategy Framework Directive by proposing food web indicators based on morphologically and habitat defined functional groups.