In this study, we develop a characterization of bacterial spore resistance to NIR pulsed light under modalities traditionally used in multiphoton microscopy. Energy dose and laser power are both key parameters in spore and bacterial cell inactivation. Surprisingly, spores and vegetative cells seem to show a similar sensitivity to pulsed NIR, spores being only 2-fold more resistant than their vegetative counterparts. This work enables us to eliminate certain hypotheses concerning the main driver of spore inactivation processes. Our findings suggest that damage leading to inactivation is mainly caused by photochemical reactions characterized by multiple possible pathways, including DNA damage or oxidation processes.