The present study aims to elucidate the microbiota of Egyptian mish cheese and milk, to isolate new strains and to study their biochemical properties relevant to their potential application in food and dairy industry. Among the isolates from Egyptian mish cheese there were two strains identified to be Lactobacillus fuchuensis DSM 14340T DSM and Lactobacillus paracasei DSM 20006 DSM as well as Lactobacillus acidophilius isolated from milk. These strains were evaluated for potential probiotic properties. Absence of haemolytic activity and antibiotic resistance is considered as a safety prerequisite for the selection of a probiotic strains. All of the strains were γ-haemolytic (i.e. no haemolysis). All of the strains were recorded highly lipolytic activity on Tributyrin agar base plates. Anti-bacterial activity of the cell-free culture supernatants (CFCS) of the potential probiotic strains (L. fuchuensis, L. paracasei and L. acidophilius) was evaluated against Salmonella enteritidis, Klebsiella pneumoniae, Aeromonas hydrophila, Proteus vulgaris, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli O157:H7, Staphylococcus aureus and Enterococcus faecalis. Supernatant culture obtained from Lb. Fuchsensis showed antimicrobial activity against all tested microorganisms except Klebsiella pneumoniae.Supernatant culture obtained from Lb. paracasei showed antimicrobial activity against all tested microorganisms except Klebsiella pneumoniae, Aeromonas hydrophila, E. coli and E. coli O 157:H7. Supernatant culture obtained from L. acidophilius showed antimicrobial activity against all tested microorganisms except Klebsiella pneumoniae, Aeromonas hydrophila, E. coli O 157:H7 and Staphylococcus aureus. The commercial antibiotics sensitivity test against the potential probiotic strains (L. fuchuensis, L. paracasei and L. acidophilius) was evaluated.