The aim of this research was to evaluate the virulence of Metarhizium anisopliae on the eggs and larvae of Rhipicephalus sanguineus, as well as the LC50 on R. sanguineus larvae, were determined and the germination (at 18 and 24 h post-inoculation) and radial growth (after 14 days) of four strains of M. anisopliae. We evaluated the pathogenicity on the eggs and larvae of all tested strains of R. sanguineus at day 3 and 7. The LC50 of four strains of M. anisopliae was determined by Probit analysis, and a chi-square test was used to determine the relationship of LC50 with variable radial growth and germination at 18 and 24 h. Germination at 18 h and 24 h ranged from 83.8–92.5% and 89.5–100%, respectively. The average radial growth ranged from 2.3–3.3 mm per day. The pathogenicity results for eggs and larvae showed 100% mortality for both biological stages. The Ma4 strain showed an outstanding LC50 value with 1.78 × 10E6 conidia mL-1. M. anisopliae is effective for controlling R. sanguineus, and that the radial growth characteristics and germination were not correlated with the fungal virulence against R. sanguineus larvae. We can conclude that there are differences between the evaluated strains of M. anisopliae in regard to germination at 18 and 24 h, radial growth and virulence against the larvae of R. sanguineus. There is also no relationship between the biological characteristics and virulence of M. anisopliae against R. microplus larvae.