AIM: To describe the antimicrobial susceptibility of mastitis-causing bacteria isolated from milk samples submitted to commercial laboratories over a period of 40 months. METHODS: The records of reported results of milk samples submitted by veterinary practitioners to five commercial veterinary laboratories in the North and South Islands of New Zealand, between August 2003 and December 2006 were reviewed. Logistic regression was used to analyse the effect of year, island, and the interaction of year and antimicrobial on the probability of antimicrobial susceptibility for each pathogen and antimicrobial combination, where the causative bacteria had >1,000 susceptibility tests in total and the antimicrobials was tested on >500 isolates. A total of 9,262 isolates were included in this study, with an average of nearly seven susceptibility tests per isolate, totalling 62,918 tests. RESULTS: Streptococcus uberis isolates demonstrated high overall susceptibility (>90.0%) to the majority of antimicrobial agents except ampicillin (81.7%), lincomycin (85.3%), trimethoprim/sulphonamide combination (88.6%), and, as expected, aminoglycosides (<4%). The susceptibility of Strep. dysgalactiae was similar to that of Strep. uberis, except for greater susceptibility to oxacillin (98.1%) and much lower susceptibility to tetracyclines (11.2%). The susceptibility of Staphylococcus aureus isolates was markedly different from that of Strep. uberis for the majority of antimicrobials tested. Susceptibility of Staph. aureus was lower than 90% to ampicillin (73.4%), erythromycin (74.7%), lincomycin (66.1%), penicillin (73.1%), and streptomycin (71.7%). No antimicrobial was effective against all Staph. aureus isolates. Minor changes were found in the overall susceptibility of the main mastitis-causing bacteria between 2003 and 2006. CONCLUSIONS: The antimicrobial agents intended for treatment of bovine mastitis currently available in New Zealand generally demonstrated good in-vitro efficacy against streptococci and staphylococci, with the exception of aminoglycosides. CLINICAL RELEVANCE: Analysis of the results of antimicrobial susceptibility of bacterial isolates from milk samples from dairy cows in New Zealand provides useful data for surveillance purposes, and a baseline for identifying changes in antimicrobial sensitivity in this population. However, the variation in antimicrobial susceptibility between individual isolates means that these data are of limited value when determining treatment of mastitis at the farm level.