Rhipicephalus (Boophilus) microplus is an ectoparasite of economic importance to cattle-rearing in tropical and subtropical countries, especially Brazil. This study aimed to assess the biological control activity of the fungus Metarhizium anisopliae in pastures at experimental farms belonging to the Unidade Regional de Pesquisa e Desenvolvimento de Pindamonhangaba (PRP) and Americana (IZ). Two areas were established in each location (treatment area vs. control area), and each contained seven animals. The treatment areas were sprayed with suspensions of the fungus M. anisopliae (IBCB 425) at a concentration of 5 x 10¹³ conidia per hectare (ha) in association with mineral oil. The control areas were sprayed only with water and mineral oil. Every two weeks, the levels of tick infestation on the animals were assessed in both the treated and the control area. The results from counting teleogynes on the cattle showed that the treatment with M. anisopliae was efficient, such that infestation remained greater in the control group in both locations evaluated. The annual mean efficacy was 36% for PRP and 48% for IZ. The application of the fungus M. anisopliae (IBCB 425) to pastures reduced the infestation of R. microplus and may constitute an effective strategy to combat this ectoparasite. Further studies will enable the refinement of this treatment.