The rise of antimicrobial resistance is reducing the effectiveness of current therapies and highlights the need for new antimicrobial agents. Marine environments, rich in microorganisms adapted to extreme conditions, represent a promising reservoir of bioactive compounds. The marine bacterium Marinomonas mediterranea secretes, among others, lysine oxidase (LodA) and glycine oxidase (GoxA), enzymes that generate hydrogen peroxide and exhibit antibacterial activity. Based on this mechanism, we hypothesized that modifying specific experimental con-ditions could enhance the antimicrobial potential of the secreted enzymes and additional com-pounds, broadening their activity spectrum. In this study, culture conditions were optimized to promote the production of inhibitory molecules, and the antimicrobial activity of the M. medi-terranea secretome was assessed against clinically relevant microorganisms. The concentrated secretome obtained from growth in Marine Broth exhibited antimicrobial activity against the Gram-negative and Gram-positive bacteria analyzed and several pathogenic fungi, an effect at-tributable to some proteins present in it. Among them, LodA and GoxA, previously associated with bacterial growth inhibition, also appear to contribute to antifungal activity. Moreover, valyl-tRNA synthetase emerged as a potential new antimicrobial factor. These findings high-light the biotechnological potential of marine microorganisms and support further exploration of their secretomes for the development of new antimicrobial agents.