Sea temperature, the best predictor of the geographic distribution of marine organisms, is, at Gray's Reef, within the tropical range during the summer but may fall below the lethal thermal minimum of many tropical fish species during the winter. Even though tropical reef fishes colonize the reef annually as drifting eggs and larvae, they are often absent during the colder winter months. The larger non-resident fishes probably migrate away from the reef in search of warmer offshore water. But many of the smaller reef residents, that cannot migrate, may not survive the low winter temperature stress.

In terms of its fish fauna, Gray's Reef can be considered intermediate or transitional. The subtropical fauna of the southeast U.S. coast includes a combination of species characteristic of the tropical western Atlantic (Caribbean) and species characteristic of the temperate/sub-tropical western north Atlantic. Its cross-shelf location between more environmentally variable (stressed), inshore habitats, and more environmentally stable, deeper, offshore habitats results in a fish community that is intermediate between lower-diversity inshore and higher--diversity offshore reefs.

The invertebrates that inhabit the exposed rock surfaces and sandy areas of Gray's Reef include bryozoans (moss animals), hydroids, ascidians (sea squirts), barnacles, tube worms, sponges, hard corals and gorgonians (sea whips and sea fans). They form an attached 'turf' of marine life that provides refuges and habitat spaces for the mobile residents of the reef. Mobile invertebrates include sea urchins, sea cucumbers, brittle stars, sea stars, snails and other mollusks, crabs, lobsters, shrimps, worms, octopods, and fishes. Nearby sandy habitats support a different sort of bottom-dwelling invertebrate community. Here, sea pens, sea pansies, sea cucumbers, sea biscuits, worms, mollusks, and crustaceans are adapted to life in soft sediments.

Drifting marine life (plankton) is abundant at Gray's Reef. The larger forms include the comb jellies, the jellyball or cabbage head, the sea nettle, or summer jellyfish, and the Portuguese man-of-war. The latter two can produce painful stings but are only rarely encountered.

Loggerhead sea turtles, which nest on all of Georgia's sea island beaches, can be found basking at the surface at the sanctuary. Divers frequently encounter them resting under the reef overhangs. Green, Kemp's ridley, and leatherback sea turtles are also known to occur along the Georgia coast.

Dolphins (spotted and bottlenose) are the most often encountered cetaceans at Gray's Reef. The endangered northern right whale is a regular visitor to Georgia coastal waters in the vicinity of Gray's Reef. The frequency of mother-calf pair sightings suggests that this area may be an important habitat for calving. As such, it is wise to stay clear of whales in this area and report any sightings to The Georgia Conservancy, Savannah, Georgia; Gray's Reef National Marine Sanctuary Headquarters, UGA Marine Extension Center, Savannah, Georgia; or the Coastal Resources Division, Georgia Department of Natural Resources, Brunswick, Georgia.

Sea birds seen in the vicinity of Gray's Reef, include petrels, shearwaters, gannets, phaleropes, jaegers, and terns. Many are migratory species and hence seasonal. The biological interaction between feeding seabirds, baitfish (surface schools of herring or scad), and pelagic predators (mackerels and tunas) is particularly evident in the vicinity of reefs.