Rookery Bay Reserve has over 50 historic sites within its boundaries. These sites are mostly prehistoric midden sites located on mangrove-forested islands and within coastal scrub and pine flatwood communities. However, there are also several sites in the Henderson Creek area that are relatively undisturbed remnants of pioneer settlements dating from the 1800s.

Some of these sites have been the subject of research projects conducted by visiting scientists in cooperation with the Florida Division of Historical Resources. Most notably, a recent study was completed in the Ten Thousand Islands that cataloged a series of prehistoric sites located within both RBNERR and the Ten Thousand Islands National Wildlife Refuge. Records for these sites are on file at the RBNERR headquarters facility. A comprehensive inventory of sites and artifacts is being developed by RBNERR staff.