The value of susceptibility tests for the selection of topical otological antimicrobial agents is unclear. Laboratories test antibiotic concentrations substantially lower than concentrations supplied in topical formulations. Additionally, microbiological consensus statements are not available for topical antimicrobials. The primary aim of this study was to measure the minimum inhibitory and bactericidal concentrations of enrofloxacin, gentamicin, marbofloxacin, neomycin, orbifloxacin, polymyxin B and silver sulfadiazine from concentrations available in otological formulations (COF) to 1:59,000 dilution. The secondary aim was to evaluate the effect of Tris-EDTA in conjunction with these antimicrobial agents. Twenty resistant clinical isolates [Staphylococcus pseudintermedius (n = 10) and Pseudomonas aeruginosa (n = 10)] were tested by broth microdilution using a concentrated inoculum (3.75 × 107 cfu/mL). Concentrations available in otological formulations were at least 26× greater than the MICs for S. pseudintermedius and P. aeruginosa. COFs of polymyxin B and SSD were 27× greater than the MBCs for P. aeruginosa, whereas all other antimicrobial COFs were equal to or less than the MBCs for both organisms. Tris-EDTA significantly reduced the MICs of all antimicrobials, except with SSD for P. aeruginosa, and it significantly increased the MIC of SSD for S. pseudintermedius. Further studies are warranted to validate the present results in vivo. COFs are inhibitory and less likely bactericidal, with few exceptions, against resistant strains of these organisms. Tris-EDTA may be advantageous for P. aeruginosa whereas no additional benefit is afforded against S. pseudintermedius. Susceptibility tests may not be useful for the selection of topical otological antimicrobial agents.