Night-time light from urban sources detects subtle urbanization patterns and assesses their ecological impact. This study examines the trends in night-time lighting across Key Biodiversity Areas (KBAs) in India, using two datasets such as the Defense Meteorological Satellite Program’s Operational Linescan System (DMSP OLS) for 1992–2013 and the Visible Infrared Imaging Radiometer Suite (VIIRS) for 2014–2020. This chapter assesses the extent of artificial lighting increase in KBAs and its potential effects on biodiversity conservation. Utilizing Google Earth Engine for data processing and the Mann–Kendall test for trend analysis, we found that 53.90% of KBAs showed significant positive trends in stable night lights from 1992 to 2013. This trend has accelerated recently, with 76.39% of KBAs showing significant positive trends in average radiance from 2014 to 2020. The regional analysis showed variations across states of India, with some displaying exceptionally high percentages of affected KBAs. The extensive increase in night-time lighting indicates increasing human encroachment and development around KBAs, possibly leading to habitat disruption, fragmentation, and variations in species behavior and interactions. The study emphasizes the need for vital conservation measures and recommends a technology–policy interface to address this concern. Recommendations include leveraging advanced remote sensing, artificial intelligence, and machine learning for improved monitoring and management of light pollution in KBAs. This chapter contributes to our understanding of human-caused pressures on biodiversity hotspots and highlights the importance of considering light pollution in conservation strategies. The findings call for instant actions to lessen the ecological impacts of artificial lighting while balancing human development needs.