Abstract Background Eravacycline is a novel synthetic fluorocycline antibacterial approved for complicated intra-abdominal infections. Objectives The purpose of this study was to assess the in vitro activities of eravacycline and comparator antibiotics against contemporary clinical isolates of Clostridioides difficile representing common ribotypes, including isolates with decreased susceptibility to metronidazole and vancomycin. Methods Clinical C. difficile strains from six common or emerging ribotypes were used to test the in vitro activities of eravacycline and comparator antibiotics (fidaxomicin, vancomycin and metronidazole) by broth microdilution. In addition, MBC experiments, time–kill kinetic studies and WGS experiments were performed. Results A total of 234 isolates were tested, including ribotypes RT001 (n = 37), RT002 (n = 41), RT014-020 (n = 39), RT027 (n = 42), RT106 (n = 38) and RT255 (n = 37). MIC50/90 values were lowest for eravacycline (≤0.0078/0.016 mg/L), followed by fidaxomicin (0.016/0.063 mg/L), metronidazole (0.25/1.0 mg/L) and vancomycin (2.0/4.0 mg/L). MBCs were lower for eravacycline compared with vancomycin for all ribotypes tested. Both vancomycin and eravacycline demonstrated bactericidal killing, including for epidemic RT027. The presence of the tetM or tetW resistance genes did not affect the MIC of eravacycline. Conclusions This study demonstrated potent in vitro activity of eravacycline against a large collection of clinical C. difficile strains that was not affected by ribotype, susceptibility to vancomycin or the presence of certain tet resistance genes. Further development of eravacycline as an antibiotic to be used in patients with Clostridioides difficile infection is warranted.