The multi-level benefits that marine organisms gain when protected from fishing are well acknowledged. Here, we investigated the effects of a 40-year trawling ban on the status of targeted and non-targeted marine species within a major fishing ground in the northeastern Mediterranean Sea (Thermaikos Gulf, Aegean Sea). Biomass and somatic length of fish and invertebrates (six commercial and three non-commercial demersal species) were measured in three areas of varying fishing pressure, depending on the temporal and spatial operational regimes of fishing vessels. The positive effects of fishing restrictions on the studied demersal stocks were clearly revealed, as the commercial fish species exhibited higher biomass in the intermediate and low pressure areas, as well as increasing maximum and mean total length (and other length indicators) with decreasing fishing effort. The mean total length of non-commercial species generally did not differ among areas, except for species caught and discarded at high rates. The present study shows that fishing does alter the population structure and biomass of commercial demersal species, and that fishing restrictions greatly contribute to improving the status of demersal populations within the restricted areas by providing a refuge for large individuals and their important contribution to the gene pool.