The work presented examines the effects of soil moisture on the virulence of selected entomopathogenic nematode isolates in the laboratory and has broad implications for managing soil-dwelling insects in all production systems. Two species of entomopathogenic nematodes, Heterorhabditis bacteriophora (Oswego and Tuscarora strains) and Steinernema glaseri (NC1 strain), were applied to sandy loam soils ranging from below the permanent wilting point of plants to near saturation. Fluctuations in soil moisture were created by simulating a rainfall or irrigation event (i.e., rehydration), or by allowing soil to dry. Nematode virulence was evaluated by measuring insect mortality in Galleria mellonella larvae bioassays. Soils prepared at four moisture contents were inoculated with nematodes and subsequently bioassayed at their original moisture contents and after rehydration, over an 18-mo period. Insect mortality increased with soil moisture content for both H. bacteriophora isolates but was highest in relatively low moisture soils (≈−15 bar) for S. glaseri. Insect mortality was generally low in low-moisture soils before rehydration but rebounded to high levels posthydration. Both isolates of H. bacteriophora nematodes were reactivated to cause high insect mortality (≥ 98%) as long as 18 mo after infective juveniles were inoculated into very low moisture soils (≈−30 bar). The posthydration insect mortality levels in the S. glaseri treatments were significantly lower than in the H. bacteriophora treatments. For all nematode treatments, the cumulative insect mortality of prehydration and posthydration bioassays was higher in lower moisture soils compared with higher moisture soils. In an additional experiment, moisture thresholds for activation of nematodes in dry soil were determined.