The tomato borer Tuta absoluta Meyrick is a serious tomato pest that has lately undergone a rapid expansion, causing severe crop losses. An integrated management is required to control this insect, within which biological control is now beginning to play a key role. In this regard, the effectiveness of a liquid formulation based on strains of the fungus Metarhizium anisopliae variety anisopliae (Metschnikoff) Sorokin (4.46 × 10 viable conidia per milliliter), applied together with irrigation water, has been evaluated by laboratory tests on different populations of T. absoluta. A bioassay method has been developed to test the efficacy of the product. The technique chosen has been validated and the different studied populations have been typified according to their susceptibility, determining the baseline susceptibility of the pest to the fungus. The results revealed a complete efficacy of M. anisopliae against pupae of T. absoluta at the recommended label rate (5.58 × 10⁹ viable conidia per liter) for the populations assayed. Moreover, a notably lower dose was also sufficiently effective to control the tomato borer populations because values of LC₉₀ lower than 3 × 10⁹ viable conidia per liter were obtained unfailingly. The most sensitive populations were those collected in Almeria and Nijar, Spain, with LC₅₀ values of 0.21 and 0.22 × 10⁹ viable conidia per liter^-1, respectively. The average value obtained for LC₅₀ was 0.34 × 10 viable conidia per liter^-1 and 2 × 10 for LC₉₀. These results show the potential of M. anisopliae to control pupae of the tomato borer in integrated pest management programs.