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In the present study, kashk samples were collected from two regions of Iran, the Fars (Abadeh) and Razavi Khorasan (Kalat) provinces. Fifteen bacteria were isolated and physiological and biochemical assays were performed. After identification to the genus level, eight isolates were identified as lactic acid bacteria (LAB) and subjected to molecular identification and probiotic properties assays. The results revealed that the isolates were <i>Enterococcus faecium</i> KKP 3772 (KF1), <i>Enterococcus faecium</i> C1 (KF2), <i>Pediococcus pentosaceus</i> H11 (KF3), <i>Pediococcus pentosaceus</i> VNK-1 (KK4), <i>Lactococcus lactis</i> RSg (KK1), <i>Enterococcus faecalis</i> P190052 (KK2), <i>Enterococcus mundtii</i> CECT972T (KK3), and <i>Lactiplantibacillus plantarum</i> PM411 (KK5). Only the numbers of <i>L. lactis</i> RSg (KK1) and <i>Lpb. Plantarum</i> PM411 (KK5) decreased to below 9 Log CFU/mL after acidic conditions (pH = 3) and showed weak antibacterial activity. <i>Enterococcus mundtii</i> CECT972T (KK3) and <i>E. faecium</i> C1(KF2) were highly susceptible to bile salts, while <i>P. pentosaceus</i> VNK-1 (KK4) and <i>P. pentosaceus</i> H11 (KF3) showed the highest resistance. All of the isolates were resistant to tetracycline and sensitive to chloramphenicol and gentamicin. The antimicrobial activity of <i>P. pentosaceus</i> VNK-1 (KK4) and <i>P. pentosaceus</i> H11 (KF3) was higher than other isolates and consequently, their inhibition zones were larger. The adhesion capabilities of LAB isolates to intestinal epithelial cells were evaluated by examining the auto-aggregation factor and cell surface hydrophobicity. The highest and lowest cell surface hydrophobicity and auto-aggregation were obtained from <i>P. pentosaceus</i> VNK-1 (KK4) and <i>E. mundtii</i> CECT972T (KK3), respectively. In general, <i>P. pentosaceus</i> VNK-1 (KK4) and <i>P. pentosaceus</i> H11 (KF3) have shown better probiotic properties as compared to other isolates.