With the progress of urbanization, the importance of biodiversity conservation and sustainable ecosystem service use in urban systems has been entirely acknowledged. We are recognizing the need to redesign city systems from the viewpoint of the relationship between urban activities and natural ecosystems. In this study, we evaluated the dependence of human activities within a large metropolitan region on ecosystem services. Our evaluation focused on the civilian sector (which consists of business and residential activities) of the municipalities of Osaka prefecture, Japan. This study applied a framework known as Ecosystem Services Use (ESU), an ecological footprint-based evaluation method, in order to identify the characteristics of Forest Ecosystem Service Use (FESU) by a municipality. FESU was estimated in hectares by integrating various statistics on human activities and using conversion functions that reflect the features of the urban systems of each municipality. The results showed that FESUCO2, the variable representing the forest area needed to compensate for carbon dioxide emissions, represented the largest portion of total FESU in all municipalities, indicating that energy consumption through urban activities plays a significant role in increasing dependence on forest ecosystems. Additionally, a cluster analysis was performed with variables related to municipality character, FESU, and external dependence. The 43 municipalities of Osaka were clustered into five groups. By applying the ESU framework, this case study demonstrates a viable method of quantitatively evaluating the dependence of urban activities on ecosystem services and helps us understand the sustainability of cities’ use of ecosystem services.