The Everglades National Park comprises several significant habitats such as extensive mangrove forests, freshwater sloughs, saltwater marshes, tropical hardwood hammocks, cypress, and pine woodlands, open sawgrass prairies, marl prairies, wet prairies, islands of Florida Bay, and seagrass ecosystems. This vast network of wetlands and forests that make up the fragile ecosystem of the National park is fed by a very shallow, slow-moving river water from Lake Okeechobee. The Park also features the largest contiguous stand of protected mangrove ecosystem in the entire Western Hemisphere.

The mosaic of important habitats that are found within the Everglades National Park supports a rich assemblage of flora and fauna that are unique to the region and are found nowhere else on Earth. Due to its location at the interface of the temperate region of North America and the tropical region of the Caribbean, the Everglades National Park hosts floral species from both the biomes. The park serves as an important habitat for a wide variety of floral species like wildflowers, broad-leafed aquatic plants, muhly grass, succulents, various scrub bushes like saw palmetto, poisonwood, wild coffee, and white indigo berry; trees such as southern live oaks, gumbo-limbo, wild tamarind, slash pines, and conifers; and epiphytes like bromeliads, orchids, ferns, and Spanish moss. Mangroves like red mangroves, black mangroves, and white mangroves are found in the Everglades National Park.

The Everglades National Park is home to a large number of faunal species. Various insects and other invertebrate animals like crustaceans, mollusks (little tree snail), spiders, centipedes, and millipedes are found here. The park provides an ideal habitat for several amphibian species like the American green tree frog and southern leopard frog. Several reptilian species like the eastern diamondback rattlesnake, eastern indigo snake, green anole, water moccasin, American alligator, American crocodile, and the endangered Atlantic green sea turtle, hawksbill turtle, Atlantic ridley turtle, Atlantic loggerhead turtle, and leatherback sea turtle are found here.

Over 300 species of fish are found in the freshwater marshes and the marine coastline of the Everglades National Park. Some of the important fish include redfish, bonefish, spotted seatrout, blue tilapia, snook, tarpon, northern red snapper, and bass.

It is believed that humans first started to inhabit the southern region of Florida up to 20,000 years ago. The region that is currently the Everglades National Park was mainly inhabited by the Indigenous Calusa peoples. Several archaeological excavations have found many architectural shellworks, earthenwares, and various other materials which provided ample evidence about the highly organized society that was maintained by the Calusa.

Human activities have led to the introduction of many invasive species in the Everglades National Park. Some of the non-native floral species that are found here include the Australian melaleuca tree, Old World climbing fern, Chinese privet, and Brazilian peppertree. The invasive faunal species include the Asiatic clam, lobate lac scale insect, bromeliad beetle, Mayan cichlid, walking catfish, Cuban tree frog, Monk parakeet, Burmese python, Nile monitor, and wild boar. To prevent new invasions and to keep the invasive species in check, the Comprehensive Everglades Restoration Plan was introduced by the US Government in 2000. In addition to these introduced plant and animal species, the Everglades National Park is also threatened by urban development, water diversion, pollution, and agricultural expansion.