The resilience of bacterial and fungal spores to common decontamination methods is a major concern in several fields including food industry. In contaminated products, those latent forms can lead to a delayed microbial development with potential hazardous health effects. This study investigates the efficiency of an ultra-high irradiance (UHI) LED device to induce inactivation of spores Bacillus cereus, Saccharomyces cerevisiae and Penicillium spp. Suspensions of bacterial and fungal vegetative cells and spores were exposed to UHI 385 and 405 nm light for periods up to 30 min. The 385-nm wavelength showed a higher potential than 405 nm with a reduction of S. cerevisiae spores of 4.2- and 0.8- log10 CFU/ml after 30 min, respectively. Similar results were observed on B. cereus endospores with a 4.2 log10 CFU/ml reduction after 30 min at 385 nm. As expected, due to the documented resistance of spores, the doses required inactivating vegetative cells of B. cereus and S. cerevisiae were significantly lower with a 4.5-log10 CFU/ml reduction achieved after 2 and 15 min at 385 nm, respectively. After a 15-min treatment, spore population of the filamentous fungi P. digitatum and P. expansum were reduced by 4 and 1.5 log10 CFU/ml, respectively. The application potential of UHI 385-nm has been assessed in a challenge test on infected mandarins. After 6 days, a reduction of mycelial and sporulation areas of 72% and 85%, respectively, has been observed on treated fruits. Great promises go along with UHI 405 and 385 nm light treatment for applications in fields where microbial resistance is a hurdle.