Extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant are characterized by the World Health Organization as pathogens for which new antibiotics are urgently needed. Omadacycline and eravacycline are two novel antibacterials within the tetracycline class. There are limited data regarding the comparison of the activities of omadacycline, eravacycline and tigecycline against isolates with different antimicrobial susceptibility profiles. Our objective was to compare the activities of omadacycline, eravacycline and tigecycline against a collection of isolates, including non-ESBL-producing, ESBL-producing and carbapenem-resistant strains. Ninety-four isolates, including 30 non-ESBL-producing, 30 ESBL-producing and 34 carbapenem-resistant (22 carrying , 12 carrying ) strains were included in the study. ESBL and carbapenemase genes were detected by conventional PCR. Omadacycline, eravacycline and tigecycline MICs were determined by the gradient diffusion method and interpreted using US Food and Drug Administration (FDA)-defined breakpoints. Overall, the percentage of tigecycline-susceptible strains (97.9 %) was higher than the percentage of omadacyline-susceptible (75.5 %) and eravacycline-susceptible (72.3 %) strains. The omadacycline and eravacycline susceptibility rates were 83.3 % among non-ESBL-producing isolates and 66.7 % among ESBL-producing isolates. The most common ESBL gene detected was (90 %), followed by (50 %) and (50 %). The omadacycline and eravacycline susceptibility rate among isolates carrying was 33.3 %, whereas it was 100 % among isolates that do not carry . The omadacycline and eravacycline susceptibility rates among carbapenem-resistant isolates were 76.5 and 67.6 %, respectively. The omadacycline susceptibility rates among -positive and -positive isolates were 77.3 and 75.0 %, respectively. The eravacycline susceptibility rates among -positive and -positive isolates were 68.2 and 66.7 %, respectively. One carbapenem-resistant isolate was intermediate and one ESBL-producing isolate was resistant to tigecycline. Overall, tigecycline was the most active tetracycline against isolates. Omadacycline and eravacycline showed excellent activity against ESBL-producing isolates that do not carry . Omadacycline showed reasonable activity against carbapenem-resistant isolates carrying or .