Freshwater species face significant challenges from direct and indirect anthropogenic impacts, resulting in a global decline in freshwater biodiversity. Protected areas are a key tool for addressing this decline, but their effectiveness in covering freshwater biodiversity remains uncertain. To investigate this, we analyzed the distribution of 182 freshwater macroinvertebrates, 26 vertebrates, and 19 macrophyte species across the Cuban biodiversity hotspot, assessing their protection coverage against the 17% and 30% conservation targets set by the Convention on Biological Diversity. Using an ensemble of four species distribution modeling techniques (Maxent, BRT, RF, SSN), we evaluated species overlap with Cuba’s current protected areas and conducted spatial conservation prioritization exercises that (i) included (lock-in) and (ii) excluded (lock-out) these protected areas. Results revealed that 41% (90 species) and 71% (161 species) failed to meet the 17% and 30% targets, respectively. Many insufficiently protected species are globally threatened or endemic to the Cuban archipelago, further heightening their extinction risk. Our findings also demonstrate that including current protected areas in conservation planning requires significantly larger areas to meet the 30% representation target, as many of these areas are redundant for the species studied. Excluding current protected areas, however, achieves conservation goals more efficiently, requiring fewer resources. Both approaches highlight the need to improve connectivity, particularly in upstream regions often neglected under the current protected area configuration. Overall, freshwater biodiversity in Cuba is poorly represented within existing protected areas. Meeting the 30% conservation target would require protecting an additional 30–70% of area, focusing on headwaters and underrepresented taxa. Expanding Cuba’s National System of Protected Areas (SNAP) to prioritize freshwater species, especially endemic ones, is essential. Spatial conservation planning that integrates lock-out and lock-in approaches can optimize resource use while enhancing connectivity across key rivers and tributaries.