Hortaea werneckii (Ascomycota) is a halotolerant black yeast, distributed primarily in hypersaline, marine and in terrestrial environments, from Atacama desert rocks to deep sea water. So far, this species was mainly studied as a biological model to understand the halotolerance mechanisms in eukaryotic cells. While some biosynthetic gene clusters seem to be present in this yeast, very few chemical investigations have been carried out, making H. werneckii an interesting candidate for marine biodiscovery. The aim of this study was to investigate the chemical diversity of 66 different strains of H. werneckii, both haploid and diploid, isolated from multiple environments. Therefore, a metabolomic study was conducted through UHPLC-HRMS/MS, in order to compare their composition and highlight some interesting chemical entities. The data were treated with Mzmine software and subjected to statistical analysis. A Feature-Based Molecular Network was then elaborated on GNPS. A strain isolated from Atlantic hydrothermal springs (Hortaea werneckii UBOCC-A-208029) was further subjected to a comprehensive study, in order to isolate and identify antimicrobial compounds. The ethyl acetate extract of this strain showed a promising activity against MRSA (IC50 value 16.2 ± 0.3 µg/mL). Fractionation was conducted by Flash chromatography and semi-preparative HPLC. Characterization of the purified compounds will be performed based on NMR and HRMS data, and their activity against MRSA will be evaluated.