This study explores the potential probiotic properties of yeasts isolated from various Chilean honeys, focusing on Ulmo, Quillay, and Mountain honeys. Six yeast strains were identified, including Zygosaccharomyces rouxii, Candida sp., Schizosaccharomyces pombe, Rhodosporidiobolus ruineniae, Clavispora lusitaniae, and Metschnikowia chrysoperlae. Phenotypic characterization involved assessing their fermentative performance, ethanol and hops resistance, and cross-resistance. Ethanol concentration emerged as a limiting factor in their fermentative performance. The probiotic potential of these yeasts was evaluated based on resistance to high temperatures, low pH, auto-aggregation capacity, survival in simulated in vitro digestion (INFOGEST method), and antimicrobial activity against pathogens like Escherichia coli, Staphylococcus aureus, and Salmonella enteritidis. Three yeasts, Zygosaccharomyces rouxii, Schizosaccharomyces pombe, and Metschnikowia chrysoperlae, exhibited potential probiotic characteristics by maintaining cell concentrations exceeding 106 CFU/mL after in vitro digestion. They demonstrated fermentative abilities and resistance to ethanol and hops, suggesting their potential as starter cultures in beer production. Despite revealing promising probiotic and technological aspects, further research is necessary to ascertain their viability in producing fermented foods. This study underscores the innovative potential of honey as a source for new probiotic microorganisms and highlights the need for comprehensive investigations into their practical applications in the food industry.