Duponchelia fovealis (Lepidoptera: Crambidae) is an invasive species that has had a large impact on strawberry crops in Brazil. Pesticides have had limited effectiveness and the use of biological control agents to improve its management is the most appropriate approach. The aim of this study was to evaluate the pathogenicity and virulence of entomopathogenic fungi—isolated from soil—against Duponchelia fovealis larvae under laboratory and greenhouse conditions. Pathogenicity screenings were performed for twenty isolates from Beauveria bassiana, Beauveria caledonica, Isaria javanica, Metarhizium anisopliae, and Lecanicillium sp. against third instar larvae of D. fovealis at the concentration of 109 conidia·mL−1. Lethal concentration (LC50) and lethal time (LT50) were determined for the most pathogenic isolates and for one commercial mycoinsecticide. Mortality rates varied from 10 to 89%. The isolates B. bassiana Bea1, Bea110, Bea111 and I. javanica Isa340 were the most pathogenic. The most virulent isolates were B. bassiana Bea111 and I. javanica Isa340 with LC50 values of 2.33 × 106 and 9.69 × 105 conidia·mL−1, respectively. Under greenhouse conditions, the efficacy of LC50 of the isolates I. javanica Isa340 and B. bassiana Bea111 were 45% and 52%, respectively. Our results indicate that these isolates are strong candidates for application in the control of D. fovealis. This study is the first evaluation of soil-borne entomopathogenic fungi against D. fovealis.