Background: Antibiotic resistance in Helicobacter pylori is steadily increasing, rendering the treatment of related gastroduodenal diseases increasingly difficult. Lefamulin is a new broad-spectrum antibacterial in the pleuromutilin class, acting by suppressing bacterial protein synthesis. It has the advantages of its unique “induced-fit” mechanism, a low frequency of spontaneous mutations, stepwise development of resistance, stability in acidic environments, and potential for additive or synergistic activity when combined with certain other antibiotics against various facultative anaerobes, including multidrug-resistant isolates. Methods: We investigated, for the first time to the best of our knowledge, the activity of lefamulin against 91 clinical H. pylori isolates from symptomatic adult patients using MIC test strips. Results: Overall, lefamulin MICs₅₀ and MICs₉₀ were 0.25 and 2 mg/L versus 4 and ≥256 mg/L for clarithromycin, and 0.75 and ≥32 mg/L, respectively, for levofloxacin. Lefamulin MICs₅₀ and MICs₉₀ were 0.5 mg/L and 4 mg/L against the 61 clarithromycin-resistant (MICs, >0.25 mg/L) isolates, 0.25 and 0.75 mg/L against the 40 levofloxacin-resistant (MICs, >1 mg/L) isolates, and 0.38 mg/L and 0.75 mg/L, respectively, against the 28 isolates resistant to both agents. Conclusions: Briefly, the new pleuromutilin antibiotic outperformed in vitro both clarithromycin and levofloxacin against H. pylori isolates. Its potential usefulness in treating H. pylori infections resistant to macrolides and fluoroquinolones, and especially those with dual resistance, justifies further investigation. However, some precautions should also be considered. The use of the novel antibiotic lefamulin may offer benefits for H. pylori eradication if our results are confirmed in subsequent studies, including clinical trials.