This study examines temporal changes in water quality of the Port Honduras Marine Reserve (PHMR), Belize. Trends in dissolved oxygen (DO), salinity, temperature and pH were analyzed from ten sites throughout PHMR for statistically significant relationships from 1998–2015. This information was complemented by field observations and an evaluation of human population changes at the local, regional and national level to gain a broad perspective of water resources. Maintaining satisfactory water quality is critical for sustaining healthy ecosystems and the communities and economies reliant upon them. PHMR represents a unique link between upland watersheds and marine ecosystems, which comprise important habitat for many aquatic species. Ecotourism activities in PHMR have become increasingly popular in recent decades and generate direct and indirect income opportunities for local communities. As a result, degradation of water quality of PHMR through human activities could have substantial ecological and economic consequences for southern Belize. Overall, the analysis of water quality revealed significant seasonal patterns, slight increasing trends in DO and salinity, but overall relatively stable water quality. These results are likely related to several factors including limited coastal development, the absence of coastal beaches and low population density in southern Belize. This study provides baseline information for future research opportunities and outlines recommendations for effective management strategies of PHMR to mitigate impacts from current and future threats to water quality.