Due to the increasing need for sustainable energy and environmental quality in urban areas, the combination of aquifer thermal energy storage (ATES) and in situ bioremediation (ISB) has drawn much attention as it can deliver an integrated contribution to fulfill both demands. Yet, little is known about the overall environmental impacts of ATES-ISB. Hence, we applied a life-cycle assessment (LCA) to evaluate the environmental performance of ATES-ISB, which is also compared with the conventional heating and cooling system plus ISB alone (CHC + ISB). Energy supply via electricity is revealed as the primary cause of the environmental impacts, contributing 61.26% impacts of ATES-ISB and 72.91% impacts of CHC + ISB. Specifically, electricity is responsible for over 95% of water use, global warming potential, acidification potential, and respiratory inorganics, whereas the production of the biological medium for bioremediation causes more than 85% of the eco- and human toxicity impacts in both cases. The overall environmental impact of ATES-ISB is two times smaller than that of CHC + ISB. Sensitivity analysis confirms the importance of electricity consumption and electron donor production to the environmental impacts in both energy supply and bioremediation. Thus, future studies and practical applications seeking possible optimization of the environmental performances of ATES-ISB are recommended to focus more on these two essential elements, e.g., electricity and electron donor, and their related parameters. With the comprehensive LCA, insight is obtained for better characterizing the crucial factors as well as the relevant direction for future optimization research of the ATES-ISB system.