Bacterial isolates obtained from swine with various clinical diseases were tested for susceptibility to tilmicosin by minimum inhibitory concentration (MIC) and Kirby-Bauer disk diffusion tests using National Committee on Clinical Laboratory Standards methodology. The tilmicosin MIC90 was < or =0.125 microg/ml for Erysiopelothrix rhusiopathiae, < or = 1 microg/ml for Haemophilus parasuis isolates, 8 microg/ml for Actinobacillus suis and Pasteurella multocida type A, 16 microg/ml for toxigenic and nontoxigenic P. multocida type D, 64 microg/ml for Bordetella bronchiseptica, and >128 microg/ml for Staphylococcus hyicus and Streptococcus suis. The results of disk diffusion testing matched well with the MIC results for each pathogen. This in vitro survey of tilmicosin activity against various swine isolates suggests that further clinical evaluation of tilmicosin in swine may be warranted for disease associated with E. rhusiopathiae, H. parasuis, and A. suis but not B. bronchiseptica, S. suis, or S. hyicus.