The antimicrobial properties of non‐thermal plasma (NTP) offer promising applications in sterilizing temperature‐sensitive materials. This study investigates how treatment parameters affect the inactivation of Bacillus subtilis spores using a surface dielectric barrier discharge powered by a nanopulse supply. This setup enables NTP exposure without direct sample contact and under controlled conditions. The effects of sample distance (3−45 mm), exposure time (5–30 min), and relative humidity (RH) (40%–90%) were evaluated. Sporicidal activity was assessed using a previously established protocol, and gas‐phase species in NTP were analyzed by Fourier‐transform infrared spectroscopy (FTIR). All parameters significantly influenced inactivation. Notably, 90% RH led to up to 4‐log greater spore reduction, highlighting the critical role of humidity in enhancing NTP efficacy.