For the isolation of probiotic cultures of Streptococcus thermophilus from dahi, we collected 120 samples from the southern regions of Punjab, Pakistan. Eleven isolates were obtained, and six were scrutinized for antibacterial activities against food-borne pathogens. The carbohydrate fermentation profile of these six strains was determined by the API50 CHL system. Additionally, these strains were amplified for their 16S rRNA regions to confirm their genotypic relationship. Furthermore, phenotypic characteristics among these strains were established by S-layer protein analysis of their cell walls by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by plasmid profiling. The outer cell wall layers of these strains have 6-14 different sizes of protein bands of 27, 34, 37, 40, 45 and 60 kDa molecular weight. Similarly, except S02FT, all strains have a single prominent plasmid of 23 kbp, whereas S02FT has an additional plasmid of 9 kbp. On the basis of this unique feature and a wide spectrum of killing patterns against pathogenic bacteria, S. thermophilus S02FT was further characterized. This culture showed an optimum antibacterial activity of 800 AU/ml at pH 5.0-5.5 and a temperature of 30-37°C. It grows well in in vitro acidic conditions and tolerates bile salt up to 2% concentration. It was resistant to nalidixic acid, ciprofloxacin, gentamicin and sulphamethoxazol, but showed intermediate behaviour to vancomycin and erythromycin.