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No direct observational evidence of sprite‐produced active radicals has been presented owing to the difficulty of observing a small event area in the nighttime mesosphere, whereas sprite chemical models have indicated that sprite discharge locally affects the atmospheric composition. We present the first observational evidence of a HO2 production above sprite‐producing thunderstorms from the coincidence of temporal‐spatial observations of HO2 spectra, sprite events, and thunderstorms by two space instruments, a submillimeter‐wave limb spectrometer and ultraviolet/visible Imager and a ground‐based very low frequency radiation lightning detection network. A total of three areas was identified with enhanced HO2 levels of approximately 1025 molecules. A chemical sprite model indicates an increase in HO2 in the considered altitude region; however, the predicted production due to a single sprite event is smaller than the observed enhancement. Our observational results suggest that sprites potentially contribute 1% of nighttime background HO2 generation at altitudes of 75–80 km globally.