Calf diarrhea is an important problem that can result in death and which leads to economic losses. Probiotics in the gastrointestinal tract can be effective for the prevention of diarrhea. In this study, some strains were isolated from traditional fermented dairy products (Shubat and Kumiss) and the feces of Holstein calves and heifers. Some probiotic properties were determined using a total of 124 isolates and Lactobacterin-TK2. Most of the isolates and Lactobacterin-TK2 were adversely affected by pH 2.0; however, they maintained their viability at pH 4.0 and 0.3% bile salt. The most effective antifungals on yeast strains were nystatin, voriconazole, and ketoconazole; however, they were resistant to itraconazole and amphotericin B. The majority of LAB strains and Lactobacterin-TK2 were susceptible to penicillin and tetracycline, whereas they were resistant to trimethoprim-sulfamethoxazole. Bacillus spp. strains were susceptible to enrofloxacin, trimethoprim-sulfamethoxazole, and gentamicin but resistant to penicillin. Also, 71% of lactobacilli have high hydrophobicity, whereas other strains have low hydrophobicity or had no hydrophobicity. Antagonistic properties of some selected strains against pathogenic bacteria were examined. All of the LABs inhibited at least one pathogen. The inhibitory effect of yeast strains on pathogens could not be determined. Then, five of the LAB strains were genotypically identified as Enterococcus faecium, one as Lactobacillus casei, and the yeast strains were identified as Saccharomyces cerevisiae and Clavispora lusitaniae. L. casei K2 and S. cerevisiae S430b were selected as superior strains. These strains are capable of being used as a new probiotic candidate following in vivo trials.