Geotrichum klebahnii, a filamentous yeast-like fungus, was isolated from a cheese sample. Several in vitro tests were carried out for its probiotic characterization. This isolate showed high survival rate of 100 ± 1.8% at low pH (pH 2) and 100 ± 0.9% at high oxbile concentration (1%) and also grew well at 37°C. It showed an auto-aggregation ability of 100.00 ± 1.5% after 20 h of incubation at 37°C, as well as 36.43 ± 0.70% and 52.13 ± 1.50% cell surface hydrophobicity with xylene and n-hexadecane, respectively. It had inhibitory activity against food-borne pathogens such as Salmonella sp., Vibrio sp. and Staphylococcus aureus. G. klebahnii produced the enzymes phytase and inulinase. It was a producer of vitamin B 12 and exoploysaccharides. It assimilated up to 29.42 ± 2.1% cholesterol after 48 h of incubation at 37°C. The organism did not produce gelatinase and DNase assay, indicating its safety as a probiotic microorganism. This is the first report of the probiotic potential of yeast like fungus, G. klebahnii.