More than 300 species of birds use the refuge for either nesting, migration stops, or wintering, enhanced by the fourteen water impoundments installed since the refuge was founded. Within the Atlantic Flyway, the refuge supports wintering snow geese, Canada geese, black ducks, green-winged teals, northern pintails, northern shovelers, American widgeons, buffleheads, red-breasted mergansers, ruddy ducks, and tundra swans. Nesting waterfowl include wood ducks, American black ducks, mallards, blue-winged teals, gadwalls, and the exotic mute swans. Grebes and loons also winter at the refuge, as do brant. Seabirds can often be seen flying just beyond the surf, including handsome northern gannets. In addition to herons and egrets, the refuge also hosts a variety of both resident and migrating songbirds, especially from late April through early May and from late August through October.

When it comes to shorebirds, Chincoteague has few equals. The number of shorebirds using the refuge ranks it fourth among sites east of the Rockies at which a census has been taken. It is second in diversity. The beach, flats, marshes, and impoundments have established the refuge as one of the most important shorebird management areas in eastern North America. So captivated was Claudia Wilds over the shorebird spectacle that every year from 1974 until 1988 she took a census from April through October, providing the refuge with invaluable data and becoming one of the world's foremost shorebird experts until her untimely death in 1997. She identified Toms Cove as second only to Delaware Bay for the availability of tiny horseshoe crab eggs in the spring, an irresistible food attraction for migrating shorebirds.

Adding to Chincoteague's importance is the presence of both endangered and threatened animal species. Both the endangered Delmarva Peninsula fox squirrel, a subspecies of the eastern fox squirrel, and peregrine falcons reside and produce young on the refuge. The squirrels were reintroduced in the refuge, which is within the animal's historic range that essentially consists of the 200-mile Delmarva peninsula, occupied by the three states of Delaware, Maryland, and Virginia. Habitat losses, however, have reduced their range by about 90 percent. In the effort to recover a sustainable squirrel population, nesting boxes have been placed in the wooded areas of the refuge where they feed.

From two to four peregrine falcon young are produced annually on a tower that provides the height that the birds look for to nest. It was there that young birds were translocated and artificially reared in 1980 and 1981 in a program that proved to be successful in introducing the bird to the refuge. In fact, over 6,000 similar introductions continent-wide helped bring the bird from near extinction and made it possible for expected removal by 1999 from the endangered species list. The flight of migrating peregrine falcons along the Atlantic Coast brings as many as 900 of these birds to the refuge, where they stop for resting and feeding before continuing their fall migration.

Piping plovers love the sandy beaches of Chincoteague. They make shallow depressions for their nests. Their sand-colored eggs and fledglings are hard to see, a defense against predators but a liability where there is wheeled or foot traffic. Off-road vehicle use and foot travel is now prohibited from March 15 to September 1 on Toms Cove Hook, one of several locations favored for nesting. Because of the extreme sensitivity to human disturbances, other nesting areas are also roped off during nesting seasons. Listed as a threatened species in 1986, plovers feed in sand and mudflats and also need undisturbed beaches for their staging prior to migration. To help insure reproductive success of this reduced species, the refuge also selectively distributes crushed shells, which the birds like to use for lining their nests. The state-endangered Wilson's plover and state-threatened least tern also nest on the Hook.

Sika deer may be seen anywhere on the drives or walks. The animals were imported to Assateague Island in the 1920s, before the refuge was established and they rapidly multiplied. Sika deer are a Japanese species that are said to have been released on the island by a Boy Scout troop that received several pairs from a large landowner on the mainland. The deer population grew massively but is now maintained at around 900 individuals by an annual hunting program.

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