We assessed physical, chemical and biological conditions of streams and rivers within the eastern margin of the Maya Mountains, Belize. All sites were located within Silk Grass Wildlife Preserve (SGWP), a 27,500-acre tract of land in Stann Creek, Belize, containing 90% mature secondary growth tropical moist forest and 10% regenerative agricultural operation. Our objectives were to: a) provide biological diversity and water quality baselines through physical, biological and chemical indicators, b) understand seasonal variation in such indicators, c) identify potential agricultural activities that may be contributing to the degradation of water resources, and d) provide guidance and recommendations on best management practices that could help mitigate the potential impacts of any the farming activities. Where possible, paired sampling locations were established so that upstream sites were located within forested areas and downstream sites were located within agricultural areas of the SGWP. In meta-sedimentary watersheds, most water quality and early biological diversity metrics did not decline significantly across paired forested-agricultural monitoring sites, suggesting that current regenerative agricultural operations are not negatively affecting freshwater ecosystems. Of notable exception, metal concentration was higher in the downstream agricultural pairs in 3 of the 4 watersheds we studied. In watersheds dominated by granitic intrusions, our results showed reduced discharge, distinctive channel morphology, sand-dominated substrates, higher wood loading, and reduced dissolved oxygen compared to the surrounding region. The one granitic agricultural reach showed significant water quality degradation when compared to its paired forested upstream site. We observed 37 fish species using eDNA and >200 aquatic macroinvertebrate species based on morphology across all sites. Our results revealed that eDNA techniques did not work well for macroinvertebrates; only 28% of the taxa found with morphology were represented in eDNA results.