Biodiversity is a broad concept that encompasses the diversity of nature, from the genetic to the habitat scale, and ensures the proper functioning of ecosystems. The Mediterranean Sea, one of the world’s most biodiverse marine basins, faces major threats, such as overexploitation of resources, pollution and climate change. Here we provide the first multi-taxa inventory of marine organisms and coastal terrestrial flora recorded in southeastern Salento (Ionian Sea, Italy), realized during the project “Biodiversity MARE Tricase”, which provided the first baseline of species living in the area. Sampling was carried out by SCUBA and free diving, fishing gears, and citizen science from 0 to 70 m. Overall, 697 taxa were found between March 2016 and October 2017, 94% of which were identified to the species level. Of these, 19 taxa represented new records for the Ionian Sea (36 additional new records had been reported in previous publications on specific groups, namely Porifera and Mollusca Heterobranchia), and two findings represented the easternmost records in the Mediterranean Sea (Helicosalpa virgula and Lampea pancerina). For eight other taxa, our findings represented the only locality in the Ionian Sea, besides the Straits of Messina. In addition to the species list, phenological events (e.g., blooms, presence of reproductive traits and behaviour) were also reported, with a focus on gelatinous plankton. Our results reveal that even for a relatively well-known area, current biodiversity knowledge may still be limited, and targeted investigations are needed to fill the gaps. Further research is needed to understand the distribution and temporal trends of Mediterranean biodiversity and to provide baseline data to identify ongoing and future changes.