Ascidians are marine invertebrates associated with diverse microbial communities, embedded in their tunic, conferring special ecological and biotechnological relevance to these model organisms used in evolutionary and developmental studies. Next-generation sequencing tools have increased the knowledge of ascidians’ associated organisms and their products, but proteomic studies are still scarce. Hence, we explored the tunic of three ascidian species using a shotgun proteomics approach. Proteins extracted from the tunic of Ciona sp., Molgula sp., and Microcosmus sp. were processed using a nano LC-MS/MS system (Ultimate 3000 liquid chromatography system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer). Raw data was searched against UniProtKB – the Universal Protein Resource Knowledgebase (Bacteria and Metazoa section) using Proteome Discoverer software. The resulting proteins were merged with a non-redundant Antimicrobial Peptides (AMPs) database and analysed with MaxQuant freeware. Overall, 337 metazoan and 106 bacterial proteins were identified being mainly involved in basal metabolism, cytoskeletal and catalytic functions. 37 AMPs were identified, most of them attributed to eukaryotic origin apart from bacteriocins. These results and the presence of “Biosynthesis of antibiotics” as one of the most highlighted pathways revealed the tunic as a very active tissue in terms of bioactive compounds production, giving insights on the interactions between host and associated organisms. Although the present work constitutes an exploratory study, the approach employed revealed high potential for high-throughput characterization and biodiscovery of the ascidians’ tunic and its microbiome.