In aquaculture, probiotic yeasts have gained particular interest because of their numerous health benefits for farmed fish. Many autochthonous yeasts have been isolated and identified from fish species with potential probiotic characteristics. In the present study, four autochthonous yeast strains were identified and characterized from the intestinal tracts of 16 healthy goldfish, Carassius auratus. Their in vitro probiotic properties were examined in terms of cell surface hydrophobicity, co-aggregation, and tolerability to different pH values and bile salt concentrations. These strains were identified by culture characters and sequence analysis of ITS (Internal Transcribed Spacer) gene regions. Four strains, namely Cutaneotrichosporon jirovecii isolate jpn01, Debaryomyces nepalensis isolate jpn02, Blastobotrys proliferans isolate jpn05, and Diutina catenulata isolate jpn06, were identified and added to the NCBI GenBank with accession numbers defined as MT584874.1, MT584873.1, MT649918.1, and MT501155.1, respectively. Results demonstrated the capability of these strains to co-aggregate with several fish-associated bacterial pathogens such as Lactococcus garvieae, Vagococcus salmoninarum, Vibrio anguillarum, Yersinia ruckeri, and Aeromonas hydrophila. Only the jpn05 strain did not co-aggregate with A. hydrophila. All identified yeast isolates could grow and tolerate low pH conditions (pH 2.0) and bile salt concentrations (up to 1.5%). Of interest, the hydrophobicity (%) of the yeast isolates was 80%, 94.0%, 80.6%, and 66.4% for jpn01, jpn02, jpn05, and jpn06 isolates, respectively. In this context, our data provide important in vitro evidence for the potential probiotic features of the yeast isolates. These strains could be considered candidate probiotic yeasts; however, their application in aquaculture nutrition necessitates further in vivo assays.