IntroductionNon-conventional yeasts (NCYs) associated with spontaneous food fermentations represent an underexplored source of microorganisms with potential probiotic and biotechnological applications. Coffee cherry fermentations harbor diverse yeast communities; however, the functional properties of indigenous NCYs remain poorly characterized. This study identified NCYs isolated from coffee cherry fermentations and evaluated their probiotic-associated functional traits, including stress tolerance, biosafety, antimicrobial activity, cytocompatibility, and effects on epithelial cell migration.MethodsFive NCY isolates were identified by ITS rDNA sequencing and characterized using in vitro assays evaluating tolerance to simulated gastrointestinal conditions, osmotic and thermal stress, cell surface hydrophobicity (CSH), autoaggregation, extracellular enzyme production, hemolytic activity, susceptibility to antifungal agents, and antimicrobial activity of neutralized cell-free supernatants (CFS). Cytocompatibility of CFS was assessed in Caco-2 cells using LDH, MTT, and Live/Dead assays, whereas epithelial cell migration was evaluated using a scratch assay.ResultsFour isolates were identified as Kurtzmaniella quercitrusa, whereas one isolate was identified as Meyerozyma caribbica. All isolates exhibited strain-dependent tolerance to simulated gastrointestinal and environmental stresses. Lev3 showed the highest tolerance to gastrointestinal conditions, with survival comparable to the reference probiotic Saccharomyces boulardii CNCM I-745 (SSb), whereas Lev10 exhibited superior tolerance to osmotic and thermal stress. CSH and autoaggregation were also strain dependent, with Lev10 and Lev5 exhibiting the most favorable adhesion-related profiles among the coffee-derived isolates. All isolates were catalase-positive and exhibited variable proteolytic activity. None showed hemolytic activity, and most were susceptible to fluconazole and nystatin. Neutralized CFS exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria while displaying low cytotoxicity toward Caco-2 cells. Although CFS did not significantly enhance scratch closure relative to the untreated control, Lev11 and Lev13 maintained epithelial wound closure comparable to the control.ConclusionCoffee cherry-derived NCYs exhibited strain-dependent probiotic-associated traits, favorable preliminary biosafety profiles, antimicrobial activity, and high cytocompatibility, supporting their potential for fermentation biotechnology and the development of next-generation probiotic or postbiotic products. Further genomic, metabolomic, and in vivo studies are required to validate their efficacy, safety, and mechanisms of action.