The combined effects of EDTA-Tris and eighteen antimicrobial agents have been evaluated in eight clinically isolated methicillin-resistant bacteria (Staphylococcus aureus, Escherichia coli, Streptococcus uberis and Streptococcus agalactiae) from bovine mastitis. The antimicrobial activity was evaluated by measuring the minimal bactericidal concentration (MBC) for the antibiotics alone or in combination with EDTA-Tris in Mueller-Hilton broth and milk. Combined use of EDTA-Tris and antibiotics potentiated or antagonized activity of antibiotics against mastitic pathogens. Milk increased the antibiotic potency of erythromycin and spiramycin on S. aureus. Culture in milk changed patterns of EDTA-Tris combinational effects compared with that in standard Mueller-Hilton broth. Combined with EDTA-Tris in milk, synergic effects were observed in colistin, dihydrostreptomycin, kanamycin, erythromycin, gentamycin, oxytetracycline, streptomycin to E. coli, Str. uberis, and Str. agalactiae. However, significant antagonistic effects of milk on antibiotic susceptibility in combination with EDTA-Tris were noted in neomycin, streptomycin, penicillin, roxithromycin, and amoxicillin. This study indicates that combination therapy of EDTA-Tris with antibiotics in bovine mastitis should be used with caution because of the possible antagonistic effects of antibiotic combination with EDTA-Tris on mastitic pathogens. In addition, antibiotic susceptibility test in combination with EDTA-Tris in milk culture condition can be benefit in search of effective treatment regimen for some antibiotic-resistant bacteria of mastitis.