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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 HO 2 production above sprite‐producing thunderstorms from the coincidence of temporal‐spatial observations of HO 2 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 HO 2 levels of approximately 10 25 molecules. A chemical sprite model indicates an increase in HO 2 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 HO 2 generation at altitudes of 75–80 km globally. Plain Language Summary: HO 2 radical, a key atmospheric oxidant in the mechanisms that influence the atmospheric composition of our planet, has been predicted to be generated by lightning‐induced upper atmospheric discharge, called sprites, from water vapor. However, there has been no observational evidence of sprite‐producing active radicals due to the difficulty in specific observations that identify these radical productions by sprite, caused by factors such as a very small molecular abundance and narrow event area in the upper atmosphere. For the first time, we present observational evidence of a rise in HO 2 amount above sprite‐producing thunderstorms by using a unique method that combined observations between HO 2 detection by a super‐high‐sensitive space spectrometer, sprite emission detection using a satellite imager and ground‐based lightning detection using expand radio frequency receivers. The amount of HO 2 generated in the sprite event area can be approximated to be 40–190 times greater than the background values. Sprite chemical model simulations reproduced HO 2 enhancement by sprite events. These pieces of evidence suggest that sprites potentially contribute about 1% increase of background level of global HO 2 generation during nighttime at altitudes of 75–80 km, which can contribute to a change in the Earth's atmospheric composition. Key Points: Clear HO 2 enhancements above sprite‐producing thunderstorms were identified by coincidence of space and ground‐based observationsEnhanced HO 2 amounts in a sprite event area were estimated to be 10 25 moleculesSprites potentially contribute about 1% of nighttime background HO 2 generations at 75–80 km globally [ABSTRACT FROM AUTHOR]