Recent studies have directed attention toward isolating lactic acid bacteria (LAB) from fermented foods due to their significant functional and probiotic properties. These bacteria play essential roles in food fermentation, preservation, and potential health benefits, making their characterization from traditional food sources of scientific interest. Two LAB isolates were obtained from traditional Iranian Koozeh cheese and characterized for their potential functional and probiotic properties. The isolates were identified using 16S rRNA gene sequencing as Lactiplantibacillus pentosus and Enterococcus faecium. Their physiological and probiotic-related properties were evaluated, including tolerance to temperature, pH, and bile salts, as well as cell surface characteristics. The isolates showed survival at 15°C and 37°C, while only E. faecium demonstrated growth at 45°C. Hydrophobicity assays revealed values of 29.08 % for E. faecium and of 63.57 % for L. pentosus, while auto-aggregation was 34.77 % and 38.98 %, respectively. Both isolates showed good tolerance to bile salt and acidic conditions, with strain-dependent differences. Enterococcus faecium maintained higher viability than Lactiplantibacillus pentosus under increasing bile concentrations and low pH. Overall, both strains demonstrated survival ability under gastrointestinal-like stress conditions, with E. faecium showing greater resistance. Additionally, both isolates exhibited co-aggregation ability against Escherichia coli (PTCC No. 1330, ATCC 8739) and were non-hemolytic. The co-aggregation percentages were 60.65% for Enterococcus faecium and 27.59% for Lactiplantibacillus pentosus. Consequently, the results suggest that both strains possess promising functional properties, indicating their potential application in food fermentation and probiotic development.