Tropical freshwater ecosystems harbor unique algal biodiversity that remains underexplored. This study presents a voucher flora of benthic diatoms from two streams of southwest Puerto Rico. 82 epilithic diatom samples, collected on 11 sampling dates, were taken from 2015 to 2016 from Rio Cupeyes and Rio Guilarte which are subjected to different degrees of anthropogenic impacts. On each sampling date, a primary (riffle) and, usually but not always, a secondary habitat (run in Cupeyes and pool in Guilarte) were sampled. The diatom samples were part of a long-term monitoring program conducted by the National Ecological Observatory Network (NEON). The two tropical streams studied have low-to-medium discharge, warm water, and medium conductivity. High-resolution Nomarski Differential Interference Contrast (DIC) light microscopy and scanning electron microscopy were employed to document the diatom flora. This research offers not only a comprehensive reference for the diatoms of Puerto Rican streams but also a valuable tool for researchers working with diatoms in neotropical streams across countries within the same ecoregion and also contributes to the broader understanding of tropical freshwater ecosystems and underscores the importance of preserving these biodiverse habitats in the face of increasingly growing anthropogenic pressures.