Yeasts play an important role in nature, and soil is one of the largest reservoirs of yeasts. Seventy-seven yeast strains were isolated from southern Chilean volcanic ash soil. These 77 strains were composed of 30 strains derived from soils used as permanent grassland, 30 strains from rotating grassland, and 17 strains from native forest (considered as the control). These strains were identified using restriction fragment length polymorphisms (PCR-RFLP) in conjunction with ITS-5.8S rDNA sequencing. Additionally, a physiological characterization of the assimilation of carbon and nitrogen and the fermentation of sugars was performed. Various features of the strains were evaluated including their growth in vinasse medium, nitrogen and phosphorus content, and lipid production. Lastly, the use of strains as biofertilizers was analyzed via the cultivation of bell pepper seedlings in soil. From the RFLP profiles, the 77 strains clustered into 10 groups. Of these, only three groups could be identified to the species level, and another was classified to the level of genus (Devaryomyces hansenii, Pichia fermentan, Kazachstania exigua, Candida sp.). All strains grew in vinasse medium. Six strains (PP1, PP4, PR4, PR10, PR27 and PR29) showed a high capacity to accumulate lipids, and three strains (PP1, PP21 and PR28) have the potential to be used as biofertilizers.