Ephemeral ponds in the Munson Sand Hills region (MSH) of Apalachicola National Forest (ANF) are an essential resource in the life cycles of a variety of amphibian species, a number of which are threatened or endangered. Various types of human activities have disturbed some of these ponds threatening their survival. Although extensive research has been done on the biology of amphibians in the ponds, little is known of the invertebrates and to what extent the water quality may be affected by human impacts. We monitored 4 ponds, representing a spectrum of sizes, natural settings, and anthropogenic disturbance, in terms of water chemistry and aquatic insect assemblages seasonally for 2 years. Pond waters were characterized by acidic pH, low ionic strength, low buffering capacity, low nutrient concentrations, and phosphorus-limiting conditions. The water quality of studied ponds was similar to those reported for natural wetlands in west-central Florida. The chemistry, as compared to a nearby sinkhole, indicated that these ponds were mainly recharged with rain and had no connectivity to groundwater. Aquatic beetles (Coleoptera), dragonflies and damselflies (Odonata), and aquatic bugs (Heteroptera) were the most diverse groups of aquatic insects recorded. Species collected included many common, predatory species adapted to exploit resources in fishless, temporary ponds. Water chemistry and aquatic insect composition showed minor spatial–temporal variations among ponds. The results of this study indicate that human disturbances have not had a significant effect on pond water quality, posing no threat to amphibian and other wildlife species, and the sampled ponds had abundant and diverse aquatic insect fauna. The aquatic insect assemblages documented in this study provide evidence that pond type and the top-down effects of aquatic insects as predators are important determinants of community structure, which is a common theme observed in temporary ponds found in other regions within temperate biomes.