Entomopathogenic fungi (EPF), such as Metarhizium spp. and Beauveria bassiana, are widely used in the biocontrol of many species of insect pests. Tobacco is an economically important crop in Guangdong Province of China, but insect pests, such as Spodoptera litura Fabricius, are a major threat to production. Here, we tested the persistence of five Metarhizium species and B. bassiana in glasshouse pot and field experiments and assessed their long-term efficacy against S. litura. We found that the colony forming units of these EPF decreased by c. 93% by 180 d in the pot soils declines tended to be exponential. In contrast, declines of c. 99% in field soils were more gradual (linear), occurring throughout the 360 d experiment. Metarhizium anisopliae Ma09 had the longest estimated half-life of 41 d, while that of B. bassiana was the shortest (9 d). Fungal density in the upper soil layer (0–5 cm) decreased rapidly and was undetectable after 150 d, whereas density was consistently greatest in the mid-layer (10–15 cm). At 180 d after inoculation, strain Ma09 elicited highest rates of mortality in S. litura. We conclude that soils in Guangdong Province are all suitable for the use of Metarhizium as a biocontrol agent, where M. anisopliae Ma09 offers greatest residual activity.