ABSTRACTMycobacterium abscessusinfections are increasing worldwide necessitating the development of new antibiotics and treatment regimens. The utility of third-generation tetracycline antibiotics was reestablished; their anti-M. abscessusactivity needs further study. The activities of omadacycline (OMC), eravacycline (ERC), tigecycline (TGC), and sarecycline (SAC) were tested against two reference strains and 193 clinicalM. abscessusisolates at different temperatures (30°C and 37°C). The minimum bactericidal concentrations (MBCs) of the four drugs were determined to distinguish between their bactericidal and bacteriostatic activities. The MICs of OMC, ERC, and TGC for the reference strains and clinical isolates were summarized and compared. OMC, ERC, and TGC exhibited a high level of bacteriostatic activity againstM. abscessus. The MICs of OMC and ERC forM. abscessremained stable, while the MICs of TGC for the isolates/strains increased with increasing temperature. Notably, the MICs of OMC forM. abscessusisolates obtained in the United States are lower than for those obtained in China.IMPORTANCEThe antimicrobial activities of four third-generation tetracycline-class drugs, omadacycline (OMC), eravacycline (ERC), tigecycline (TGC), and sarecycline (SAC), were determined for 193M. abscessusisolates. The activities of the four drugs at two different temperatures (30°C and 37°C) were also tested. OMC, ERC, and TGC exhibited significant activity againstM. abscessus. The anti-M. abscessusactivity of TGC increased when the temperature was increased from 30°C to 37°C; the activities of OMC and ERC, on the other hand, remained the same. We found thatin vitroMICs of OMC against Chinese and American isolates were distinct. Evaluations inin vivomodels ofM. abscessusdisease or in the clinical setting will provide more accurate insight into potency of OMC against distinct isolates.