Antimicrobials are commonly used to treat mycoplasmosis in animals. In spite of this and the fact that antimicrobial resistance has been recorded for this group of bacteria there are no universally accepted in vitro means of testing for this resistance, nor is resistance testing for mycoplasmas a routine in most veterinary laboratories. So prior to testing for resistance to a number of mycoplasmas isolated from animals in South Africa it was necessary to compare different tests including broth and agar microdilution tests to find out which one would perform best. Using the field strains M. bovis, M. crocodyli, M. felis, M. gallisepticum and M. synoviae, and the reference strains M. gallisepticum 56USDA, M. gallisepticum VaxSafe MG vaccine strain, M. mycoides T1/44 vaccine strain, and M. mycoides Ygoat (11706) broth- and agar-microdilution minimum inhibitory concentration (MIC)tests were performed using either modified Hayflicks or Mycoplasma synoviae media. Two different metabolism indicator systems were compared in the broth microdilution test (BrMIC) namely sugar fermentation (glucose or pyruvate) with phenol red (SFS) and evidence of reduction with resazurin (AlamurBlue®). It was also tested whether amoxicillin and clavulanic acid (ACA) could be used in the tests to reduce problems associated with contamination. Statistical analyses of the tests (repeatability and linear association) indicated that the BrMIC with SFS was the most reproducible method (pooled standard deviation = 0.14). The antimicrobial ACA was found to not to affect the MIC values (R2= 0.976 to 0.996). Furthermore one hundred forty two field strains including 93 M. bovis, 5 M. synoviae, 21 M. gallisepticum, 13 M. bovirhinis, 8 M. crocodyli and 6 M. felis were tested using the BrMIC+SFS with ACA method. Generally the mycoplasmas originating from poultry were resistant to commonly used antimicrobials and had higher MIC50 and MIC90 values than isolates originating from cattle, crocodiles and cats. It was found that most of the mycoplasmas were susceptible to doxycycline (tetracycline) and enrofloxacin with the exception of M. gallisepticum where 17.9% of strains were resistant to both. Resistance to tiamulin (100%) and tylosin (20 to 64%) was high for the poultry mycoplasmas. Most field isolates tested were resistant to erythromycin, nalidixic acid, florfenicol, norfloxacin, neomycin, sulphamethoxazole, trimethoprim and sulphamethoxazole/ trimethoprim combination, mostly resistant to norfloxacin and florfenicol. It is concluded that BrMIC+SFS with ACA method is a reproducible method that reduces any problems with bacterial contamination. As observed with the poultry strains, it is quite clear that antimicrobial resistance is developing to commonly used antimicrobials such as tylosin, the related pleuromutilins, fluoroquinolones and tetracyclines. In species where antimicrobial therapy is applied routinely such as poultry and possibly feedlot cattle, it is recommended that MIC testing is done prior to any therapeutic interventions.