IntroductionPhytopathogenic fungi are a considerable concern for agriculture, as they can threaten the productivity of several crops worldwide. Meanwhile, natural microbial products are acknowledged to play an important role in modern agriculture as they comprehend a safer alternative to synthetic pesticides. Bacterial strains from underexplored environments are a promising source of bioactive metabolites.MethodsWe applied the OSMAC (One Strain, Many Compounds) cultivation approach, in vitro bioassays, and metabolo-genomics analyses to investigate the biochemical potential ofPseudomonassp. So3.2b, a strain isolated from Antarctica. Crude extracts from OSMAC were analyzed through HPLC-QTOF-MS/MS, molecular networking, and annotation. The antifungal potential of the extracts was confirmed againstRhizoctonia solanistrains. Moreover, the whole-genome sequence was studied for biosynthetic gene clusters (BGCs) identification and phylogenetic comparison.Results and DiscussionMolecular networking revealed that metabolite synthesis has growth media specificity, and it was reflected in bioassays results against R. solani. Bananamides, rhamnolipids, and butenolides-like molecules were annotated from the metabolome, and chemical novelty was also suggested by several unidentified compounds. Additionally, genome mining confirmed a wide variety of BGCs present in this strain, with low to no similarity with known molecules. An NRPS-encoding BGC was identified as responsible for producing the banamides-like molecules, while phylogenetic analysis demonstrated a close relationship with other rhizosphere bacteria. Therefore, by combining -omics approaches andin vitrobioassays, our study demonstrates thatPseudomonassp. So3.2b has potential application to agriculture as a source of bioactive metabolites.