Road–stream crossings are widespread human-built structures that can influence river connectivity and aquatic ecosystem health by altering habitat conditions and water quality. This study evaluated the conservation status of stream crossings and their relationship with aquatic macroinvertebrate abundance in southern Puerto Rico, between the municipalities of Cayey and Salinas. Fifteen crossings were assessed over one year using field-based habitat and infrastructure condition surveys, physicochemical water quality measurements, and macroinvertebrate community sampling. Crossings were classified along a conservation gradient from optimal to degraded conditions. Results showed that stream crossings in optimal conservation status supported significantly higher macroinvertebrate abundance and more diverse assemblages compared with crossings in poorer condition. Physicochemical parameters also indicated improved environmental quality at well-preserved sites, suggesting that habitat integrity promotes healthier aquatic communities. These findings emphasize the ecological importance of maintaining and restoring road–stream crossing conditions as part of sustainable infrastructure management to enhance freshwater biodiversity and ecosystem resilience in Puerto Rico and other tropical island watersheds.