Karst aquifers occur worldwide and exhibit groundwater flow responses that differ considerably from aquifers lacking fractures, bedding planes, and other karst conduits where significant and rapid groundwater flow can occur. The regional, karst Floridan aquifer system underlies the United States (US) Southeastern Coastal Plain Physiographic Region and exhibits hydrologic interconnections with overlying surficial aquifers and throughout other zones of the aquifer system, as is characteristic of other karst aquifer systems. Anthropogenic groundwater declines in this regional karst aquifer system have been documented in published literature for decades, but the impacts of those declines in this coastal plain region and the embedded ecosystems that provide essential and critical habitat for native, endemic, and federally endangered and threatened species have not been considered previously. Those anthropogenic groundwater declines reduce surfacewater levels and flows due to the capture of both groundwater and overland flow of surfacewater, resulting in induced recharge through semi-confining zones and interbasin flow through fractures and other karst conduits. This case study identifies examples from the Greater Okefenokee Swamp Basin study area and comparison areas of how those declines result in loss of historic base flow to surface waters and other capture of surface waters, ultimately increasing saltwater intrusion. Those results alter and degrade the physical, chemical, and biological integrity of the nation’s waters, in violation of the US Clean Water Act (CWA) of 1972. Historic groundwater declines from mining and other anthropogenic groundwater withdrawals from this regional karst aquifer system already threaten the survival and recovery of federally endangered and threatened species, as well as existing and proposed critical habitat for those species within this regional extent, in violation of the Endangered Species Act (ESA) of 1973. This case study and its companion publication (Part 2) appear to be the first to provide scientific support for this regional karst aquifer system as the unifying factor in habitat responses to irreversible groundwater impacts on aquatic and marine ecosystems. These adverse impacts strongly suggest that the extent of the regional Floridan aquifer system should be designated as the Southeastern Coastal Plain Ecoregion for the purpose of managing natural resources. Mining activities continue to expand in our study area, which is the Greater Okefenokee Swamp Basin. Despite that fact, no comprehensive Areawide Environmental Impact Statement (AEIS), similar to the AEIS required for phosphate mining within the Central Florida Phosphate District (CFPD) approximately a decade ago has been conducted for any of the numerous mining projects that are occurring and are proposed within the Greater Okefenokee Swamp Basin. This case study also provides examples of why a comprehensive AEIS is essential to consider all of the adverse direct, indirect, and cumulative impacts of those mining activities to the CWA, the ESA, and the irreversible losses to local economies, because federal agencies responsible for considering those adverse impacts rely on public comments to identify those adverse impacts. The mining activities authorized throughout the regional Floridan aquifer system under Category 44 Nationwide Permits (NWP) result in the same type of adverse impacts as the mining activities evaluated under Individual Permits in that region. Therefore, those Category 44 NWP mining activities also should be required to obtain Individual Permits and be evaluated under an AEIS in the Greater Okefenokee Swamp Basin. This case study also describes how Florida’s assumption of the CWA Section 404 regulatory authority in 2020 severs four sub-basins within the Greater Okefenokee Swamp Basin study area at the state line between Florida and Georgia.