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The aim of this work was to assess the virulence of strain M379 of the fungus, Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) after different passages through a suitable host and at different concentrations for the control of both acaricide-susceptible and resistant strains of the tick, Rhipicephalus (formerly Boophilus) microplus Canestrini (Ixodida: Ixodidae) in vitro. The highest value of LC50 for the susceptible strain corresponded to zero passage with 7.68 × 107 conidia/ml followed by the fourth passage with 2.68 × 107, which reduced 2.87-fold the lethal concentration. When comparing LC50 values of the fourth vs. the seventh passage (2.59 × 105 conidia/ml), the lethal concentration was reduced 103.47-fold by the seventh passage. In addition, in the resistant strain the LC50 highest value corresponded to zero passage with 4.95 × 107 conidia/ml followed by the fourth passage with 7.86 × 106, which reduced 6.30-fold the lethal concentration. When comparing LC50 values of the fourth vs. the seventh passage (1.04 × 105 conidia/ml) in the resistant strain, the lethal concentration was reduced 75.58-fold by the seventh passage. These results suggest that the number of passages on M. anisopliae through a suitable host increased its virulence on both R. microplus strains. When comparing LC50 of the zero passage through a suitable host of both acaricide-susceptible and resistant strains, the highest LC50 values corresponded to the susceptible strain with 7.68 × 107 conidia/ml followed by the resistant one with 4.95 × 107, showing that on the resistant strain the lethal concentration is reduced by 1.55-fold. When comparing the fourth passage, the highest values of LC50 corresponded to the susceptible strain with 2.68 × 107 conidia/ml followed by the resistant one with 7.86 × 106 conidia/ml, showing for the resistant strain a 3.41-fold reduced lethal concentration. Moreover, when comparing the seventh passages, the highest values of LC50 corresponded to the susceptible strain with 2.59 × 105 followed by the resistant with 1.04 × 105 conidia/ml, revealing for the resistant strain a 2.49-fold reduced lethal concentration. These results suggest that the resistant strain needs a lower concentration of conidia than the susceptible strain. In this case, the acaricide-resistant strain is more susceptible to M. anisopliae of zero- and seven-passage strains.