Laboratory experiments were done to measure the susceptibility of larvae and adults of the onion maggot, Delia antiqua (Meigen) (Diptera: Muscidae: Anthomyiidae) to 27 isolates of entomopathogenic fungi from four genera [Beauveria Vuillemin, Lecanicillium (Petch) Zare & W. Gams, Metarhizium Sorokin, and Paecilomyces Bainier]. A novel bioassay was developed for D. antiqua larvae by using a diet based on mixed vegetable powder. When evaluated in a virulence screen, the fungal isolates caused less mortality of D. antiqua larvae than adults. Only three isolates caused >50% mortality of larvae, whereas 12 isolates caused >50% mortality of adults. Fungal species was a statistically significant factor affecting the mortality of larvae but not of adults. The fungal isolates causing the most mortality of larvae tended to belong to Metarhizium anisopliae (Metschnikoff) Sorokin. Two M. anisopliae isolates (389.93 and 392.93) were evaluated in dose–response bioassays. The median lethal concentrations of the isolates against larvae were 6.1 × 10⁷ conidia ml⁻¹ for isolate 389.93 and 7.6 × 10⁷ conidia ml⁻¹ for isolate 392.93. The emergence of adult flies from pupae was reduced at high concentrations of conidia (3.0 × 10⁸ and 1.0 × 10⁸ conidia ml⁻¹). The median lethal concentrations of the isolates against adults were 1.7 × 10⁷ and 4.0 × 10⁷ conidia ml⁻¹, respectively. Some of the fungal isolates examined may have potential as biological control agents of larvae of D. antiqua and related species.