Abstract <p>Non-thermal plasma (NTP) generates a complex mixture of reactive oxygen and nitrogen species (RONS) that can impose strong oxidative stress on eukaryotic cells. While the antimicrobial potential of NTP has been widely explored, much less is known about how eukaryotic cells respond to prolonged NTP-induced stress at the cellular and molecular level. Here, we investigated the cellular effects of extended NTP exposure using the fission yeast <i>Schizosaccharomyces pombe</i> as a non-pathogenic eukaryotic model. Our results indicate that extended exposure to NTP significantly reduces cell viability and is associated with increased oxidative stress, as evidenced by increased levels of intracellular RONS and mitochondrial superoxide. These oxidative changes were accompanied by pronounced cellular responses including tubulin depolymerisation, cell cycle arrest, and impaired cell division. In contrast, no significant changes were detected in the expression of genes involved in oxidative stress response and DNA repair. The observed effects are based on cellular, phenotypic, and transcriptomic analyses, while direct identification of oxidatively modified proteins remains to be addressed in future studies.</p> Key points <p>• <i>NTP increases intracellular RONS and mitochondrial superoxide levels</i></p> <p>• <i>NTP causes tubulin depolymerisation, which is associated with cell cycle arrest</i></p> <p>• <i>NTP alters the expression of genes involved in post-transcriptional regulation</i></p> Graphical Abstract <p></p>