A recently described mycoplasma, Mycoplasma alligatoris, was isolated from dead American alligators (Alligator mississippiensis) that had demonstrated clinical signs of lethargy, anorexia, bilateral ocular discharge, edema, paraparesis, and polyarthritis. The in vitro minimum inhibitory concentration for nine antibacterial agents was determined through serial dilution in broth and plate culture for M. alligatoris isolates. The inhibitory concentration obtained for doxycycline, enrofloxacin, sarafloxacin, oxytetracycline, tilmicosin, and tylosin (<1 μg/ml) was lower than that of clindamycin (1–8 μg/ml), chloramphenicol (8–16 μg/ml), and erythromycin (32–138 μg/ml).