Background and Aim:Mastitis, primarily caused by intramammary bacterial infection, is the most expensive disease in the global dairy industry due to its negative impact on milk composition and manufacturing properties. This study aimed to evaluate the efficacy of parenteral amoxicillin in the treatment of clinical and subclinical mastitis in smallholder dairy farms in Northern Thailand.Materials and Methods:A total of 51 cows with clinical and subclinical mastitis from dairy cooperatives in Lamphun and Chiang Mai provinces, Northern Thailand, were enrolled in this study. Conventional bacteriological procedures were applied to identify the causative bacteria in milk samples from these cows before and 7 days after treatment, and antibiotic susceptibility tests were conducted using the disk diffusion method for all bacteria isolated before treatment. All cows with mastitis were administered 15 mg/kg of amoxicillin (LONGAMOX®, Syva Laboratories SA, Spain) intramuscularly every other day for 3 days.Results:Environmental streptococcal bacteria (Streptococcus uberisandStreptococcusspp.) were commonly isolated from infected quarters and were highly susceptible to amoxicillin (100%). The clinical efficacy of amoxicillin treatment for clinical mastitis cases was 80.43%, and the bacteriological efficacy was 47.82%, with opportunistic staphylococcal bacteria (coagulase-negative staphylococci) and contagious streptococcal bacteria (Streptococcus agalactiae) being the most sensitive microorganisms (100%). In subclinical mastitis cases, the bacteriological efficacy of parenteral amoxicillin was 70.45%, with environmental streptococcal bacteria (S. uberis) being the most (100%) sensitive microorganisms.Conclusion:Amoxicillin is highly efficacious and can be used to treat clinical and subclinical mastitis in dairy cows, particularly mastitis caused by environmentalStreptococcusspp. These findings could be used to guide treatment regimens in veterinary practice in smallholder dairy farms in Thailand.