In this study, the existence of intra-specific and inter-generic fungicidal activity in Xanthophyllomyces dendrorhous and Phaffia rhodozyma strains isolated from different regions of the earth was examined. Assays were performed under several culture conditions, showing that all the analyzed X. dendrorhous and P. rhodozyma strains have killing activity against Kloeckera apiculata, Rhodotorula sloffiae, and R. minuta. This activity was greater in rich media at a pH from 4.6 to 5.0. Extracellular protein extracts with fungicidal activity were obtained from cultures of all strains, and their characterization suggested that a protein of ~33 kDa is the antifungal factor. According to peptide mass fingerprinting and an analysis of the results with the MASCOT search engine, this protein was identified as an aspartic protease. Additionally, extrachromosomal double-stranded DNA elements (dsDNAs) were observed in all X. dendrorhous and P. rhodozyma strains. Although there is a high variability, two dsDNAs of 5.4 and 6.8 kb are present in all strains.