Streptococcus agalactiae is a major contagious pathogen causing bovine sub-clinical mastitis. The present investigation was carried out to determine some phenotypic characteristics of the S. agalactiae strains isolated from bovine mastitis cases in dairy cows of Shahrekord in the west-center of Iran. One hundred eighty California mastitis test (CMT) positive milk samples were bacteriologically studied. A total of 31 (17.2%) S. agalactiae isolated. Twenty eight (90.3%) of the isolates were biofilm producers. This finding may indicate the high potential of pathogenicity in isolated strains. Sixteen (51.6%) isolates were α hemolysin producers. Only 19.3%, 22.5% and 29.0% of the isolates were sensitive to streptomycin, flumequine and kanamycin, respectively. None of these three agents is recommended for treatment of mastitis cases.