This study was carried out to treatment test for bovine mastitis by the determination of adenosine triphosphate (ATP) based on firefly bioluminescence. In the susceptibility test, cephalothin which looks the most effective were sensitive to Staphylococcus sp. (72.3 %), Micrococcus sp. (84.2 %), Streptococcus sp. (72.7 %) and Gram positive bacilli (72.7 %), Gram negative bacilli were sensitive to gentamycin (92.3 %) and Yeast-like-fungi was the most sensitive to clotrimazole, and nystatin in order. When the number of bacteria, such as Staphylococcus aureus, Candida tropicalis isolated from the mastitis milk were counted by conventional agar plating technique, and compared with the concentration of bacterial ATP, it gave a good linear relationship. The content of ATP per Staphylococcus aureus, cell was 3.1fM and Candida tropicalis showed the high level of ATP (90fM). The ATP assay was applied to the determination of minimal inhibitory concentration (MIC) of various antibiotics. When Staphylococcus aureus was incubated in the presence of different concentration of tetracycline, erythromycin, kanamycin and streptomycin sulfate and the growth was monitored by the conventional agar plating technique and ATP assay, both methods showed the same results that they were 1mcg/ml, 2mcg/ml, 6.25mcg/ml and 8mcg/ml, respectively. For the determination of susceptibility of sensitive and resistant Staphylococcus aureus isolated for the milk with mastitis to tetracycline, erythromycin, kanamycin and strepomycin sulfate, the minimum time required for the test was determined by the assay of ATP every 30 minutes during incubation of 3 hours at 37deg C. ATP concentration time curve calculated on both resistant and sensitive strains incubated 3 hours as the optimum time for the determination of susceptibilities of various antibiotics examed. The ATP concentration of each test brith (antibiotic contraining), expressed as a percentage of its own control brith (antibiotic-free) indicated values of 30 % to be indicative of each antibiotic sensitivity.