Freshwater taxa are amongst the most threatened on Earth due to multiple human-induced impacts on inland waters. There is particular concern about the integrity of the freshwater biota in the Neotropical region, which is one of the most biodiverse in the world. Here, I contributed to addressing this knowledge gap by investigating multi-faceted freshwater diversity change in two Neotropical systems: Trinidad’s Northern Range, in the Caribbean, and central Mexico. First, I evaluated the conservation status of the Northern Range taxa by examining a long-term time series of diatom, invertebrate and fish diversity. Using multiple indicators, I detected metacommunity-level stability in the diatoms, alongside mixed patterns for the invertebrates and fish. Next, I investigated the signature of recreational use on these taxa by comparing trends of change in sites popular for recreation and in comparatively unperturbed sites. Contrasting trends for the three groups suggest that different organisms respond differently to river recreation. In central Mexico, I asked how species introductions and extirpations have modified fish diversity using a contemporary survey benchmarked against historical reconstructions of the native fish occurrences in the area. I revealed that indices of trait diversity are particularly sensitive to the compositional changes that have occurred, that different introduction pathways select for different fish traits, and that extirpations might be eroding unique ecological roles performed by native species. My research highlights the complexity of freshwater diversity change and the different stages of degradation exhibited by Neotropical ecosystems. I find more evidence of ecological integrity in Trinidad than in central Mexico. As my work makes clear, examining different facets of diversity in an integrated analysis can provide increasing knowledge about these less studied regions. Moreover, while the multifaceted nature of trends poses challenges for communicating conservation messages, my findings should help prioritise actions to protect these biotas.