The purpose of this retrospective study was to assess the efficacy of antimicrobial therapy for bovine acute Klebsiella pneumoniae mastitis. We evaluated data from cattle in Ehime, Japan, with naturally occurring acute mastitis due to K. pneumoniae (n=208) or Escherichia coli (n=201). Survival was significantly shorter in cattle with acute K. pneumoniae mastitis (median, 76 days) compared with the disease caused by E. coli (median 464 days). In 2004-2008, because both species were highly susceptible to cefazolin, cases of K. pneumoniae and E. coli mastitis were treated solely with cefazolin, yielding clinical cure rates of 52.8% for K. pneumoniae and 86.0% for E. coli. However, since 2009, the efficacy of treatment of K. pneumoniae mastitis with cefazolin alone has decreased. When cefazolin administered on the first disease day led to clinical improvement, treatment with cefazolin was continued. However, when cefazolin administered on the first disease day failed to yield clinical improvement, the antibiotic was switched to a fluoroquinolone on the second day, resulting in cure rates of 76.7% for K. pneumoniae and 80.0% for E. coli. These findings suggest that, when the first-line drug (e.g., cefazolin) is ineffective, promptly changing to a second-line drug (e.g., a fluoroquinolone) increases the cure rate for bovine K. pneumoniae mastitis.