Stenotrophomonas maltophilia is increasingly recognized as a nosocomial bacterial pathogen with a multi-drug resistance profile. In this study, the novel drug gepotidacin, the first compound of the novel triazaacenaphthylene topoisomerase inhibitor antibiotics class, was evaluated on its activity against clinical S. maltophilia isolates. Ninety-nine S. maltophilia isolates plus reference strain K279a (N = 100) were tested on their susceptibility towards gepotidacin in a broth microdilution. Additional susceptibility testing was performed towards the commonly applied combination trimethoprim/sulfamethoxazole (TMP/SXT), moxifloxacin, and levofloxacin. The time-kill kinetic of gepotidacin was observed in a time-kill assay. The greater wax moth Galleria mellonella was used to determine the activity of gepotidacin against S. maltophilia in vivo. Gepotidacin showed minimum inhibitory concentrations (MICs) between 0.25 and 16 mg/L (MIC50: 2 mg/L; MIC90: 8 mg/L), independently of its susceptibility towards TMP/SXT. The five TMP/SXT resistant strains exhibited gepotidacin MICs from 1 to 4 mg/L. The S. maltophilia strains resistant to the assessed fluoroquinolones showed in parts high MICs of gepotidacin. The time-kill assay revealed a time- and strain-dependent killing effect of gepotidacin. In vivo, injection of gepotidacin increased the survival rate of the larvae from 61 % to 90 % after 2 days. This study showed antimicrobial effects of gepotidacin towards S. maltophilia.