The JEM-EUSO (Joint Experiment Missions for the Extreme Universe Space Observatory) program aims at developing Ultra-Violet (UV) fluorescence telescopes for efficient detections of Extensive Air Showers (EASs) induced by Ultra-High Energy Cosmic Rays (UHECRs) from satellite orbit. In order to demonstrate key technologies for JEM-EUSO, we constructed the EUSO-Balloon instrument that consists of a ∼1 m2 refractive telescope with two Fresnel lenses and an array of multi-anode photo-multiplier tubes at the focus. Distinguishing it from the former balloon-borne experiments, EUSO-Balloon has the capabilities of single photon counting with a gate time of 2.3 μs and of imaging with a total of 2304 pixels. As a pathfinder mission, the instrument was launched for an 8 h stratospheric flight on a moonless night in August 2014 over Timmins, Canada. In this work, we analyze the count rates over ∼2.5 h intervals. The measurements are of diffuse light, e.g. of airglow emission, back-scattered from the Earth's atmosphere as well as artificial light sources. Count rates from such diffuse light are a background for EAS detections in future missions and relevant factor for the analysis of EAS events. We also obtain the geographical distribution of the count rates over a ∼780 km2 area along the balloon trajectory. In developed areas, light sources such as the airport, mines, and factories are clearly identified. This demonstrates the correct location of signals that will be required for the EAS analysis in future missions. Although a precise determination of count rates is relevant for the existing instruments, the absolute intensity of diffuse light is deduced for the limited conditions by assuming spectra models and considering simulations of the instrument response. Based on the study of diffuse light by EUSO-Balloon, we also discuss the implications for coming pathfinders and future space-based UHECR observation missions.