The Apalachicola National Estuarine Research Reserve (ANERR) conducted a system-wide biological monitoring program to describe long-term, temporal and spatial characteristics of the nekton communities found in Apalachicola Bay, Florida, USA. In a 13 year study, from July 2000 to December 2012, post-larval and juvenile fishes and macroinvertebrates (nekton) were sampled monthly using a modified trawl net to characterize seasonal, spatial (site specific) and abiotic factors, that influence the communities found in the Apalachicola Bay system. Time of year, bay salinity, which was significantly affected by flow from the Apalachicola River, and bottom type were all important factors determining which species and relative abundances make up the community structure in this system. Seasonal changes in water temperature were indicative of winter and spring recruitment of juveniles into the estuary followed by settlement in the summer and fall into different habitats, based largely upon salinity and bottom-type. Spatial differences in community structure were characterized by high abundance, and lower species richness in the northern, lower salinity portion of the estuary, and changing inversely to lower abundance and higher species richness in the southern seagrass habitats.