Background: Mastitis is one of the most devastating conditions for the dairy farms because of its alarming impact on production. Khulna is one of the dairy intensive regions of Bangladesh but comprehensive epidemiological studies regarding prevalence and risk factors of sub-clinical mastitis with antibiogram of the causative bacteria are scant. Therefore, an epidemiological study was conducted in Khulna district to investigate the prevalence and risk factor of sub-clinical mastitis in lactating dairy cows with antibiogram of the causative bacteria. Methods: Five dairy farms were selected and a total of 400 quarter milk samples of 100 dairy cows were subjected to California Mastitis Test to detect sub-clinical mastitis. Antibiogram study was performed to determine sensitivity and resistant pattern of the isolated bacteria. Results: An overall cow level prevalence of sub-clinical mastitis were recorded as 28%. Risk factors like type of breed, body condition score, milk yield, grass feeding, udder washing before milking, drying of the udder after washing, production system, dry cow therapy, stimuli before milking and milking techniques were statistically significant for the occurrence of sub-clinical mastitis. Antibiogram study revealed that most of the isolated 33.03% E. coli and 16.96% Staphylococcus sp. were sensitive to amoxicillin, gentamicin and trimethoprim/sulphamethoxazole.