Water quality in Galápagos has been deteriorating by increased human impacts over the past few decades. Water quality is a key environmental component and issue in need to be evaluated in the Pelican Bay Watershed, the biggest urban and economic development of Santa Cruz Island, for better management and regulation of water resources. This study assesses coastal and ground water bodies of Pelican Bay by employing a 9-year dataset obtained during a local water quality monitoring program conducted by the Galápagos National Park. Physical-chemical and microbial parameters were evaluated with respect to national and international water quality standards. A statistical integrated approach was performed to calculate environmental background levels of water quality parameters and to explore their seasonal and spatial variation. In addition, a sensitivity analysis was conducted to evaluate the impact of changes in tourism and residents in San Cruz Island in the degradation of water sources. Results highlighted are: (a) water is not suitable for drinking and domestic use at some inland sites; (b) saline water is used for irrigation in the highlands; (c) the presence of parameters of concern at coastal sites represent a risk for human and ecosystem health; (d) background levels may serve for defining site-specific limits to control water quality, and; (e) the influence of population change on water quality conditions varied at each site with a higher effect at coastal sites relatively to inland sites. This study provided valuable information of the water quality status in Santa Cruz Island and can serve as a baseline for effective water management and control of pollution.