The Laguna del Tigre National Park—a critical reservoir of wetland and tropical dry forest habitats—is currently threatened by human encroachment, out-of-control fires, and hydrocarbon exploitation. This rapid assessment of the region reveals a freshwater reef, two new species of fish, and range extensions for an endemic deer mouse and an aquatic snake, as well as providing detailed observations of the endangered Morelet’s crocodile and the first species list for ants and phytoplankton in the park. Of special significance are toxological studies of DNA damage to two fish species, drawing international attention to the potential effects of hydrocardon operations on the park’s ecosystems.