This study applied a guided culturomics workflow to isolate and characterize gut-associated yeasts as probiotic candidates. Culture conditions were optimized using Dixon agar and Modified Schädler Agar with a moderate antibiotic cocktail (colistin, chloramphenicol, vancomycin) to suppress bacteria without impairing yeast growth, combined with incubation at 37 °C under anaerobic conditions to mimic the intestinal environment. From three healthy donors, 305 isolates were recovered (MSA: 193; Dixon: 112). After excluding pseudohyphal morphotypes and PCR positive Candida isolates, 127 non-Candida strains remained. Safety screening (hemolysis, DNase, coagulase) reduced the pool to 26 safe isolates. Simulated gastrointestinal stress tests showed that 20 out of 26 exhibited at least 50 percent survival under pH 2.0 or 0.5% bile salts. Functional assays revealed strain-specific antimicrobial activity against Staphylococcus aureus, Escherichia coli including O157:H7 and Salmonella species, with several isolates (Y6, Y22, Y42, Y48, Y55, Y56, Y73, Y105, Y127) showing broad and strong inhibition. Two isolates Y44 and Y55 further demonstrated robust bile and acid tolerance (>50% survivability) in both conditions. All isolates displayed intracellular but not extracellular bile salt hydrolase activity, indicating a viability dependent cholesterol lowering potential. Overall, the workflow minimized bacterial interference while enriching for safe and functional yeasts.