ABSTRACT The present study evaluated the pathogenicity of Metarhizium anisopliae sensu lato (s.l.) in aqueous and oil formulations against Rhipicephalus microplus in semi-field conditions and the persistence of this microorganism in the soil. Engorged females were used in a biological assay, set in vessels cultivated with Urochloa brizantha and sprayed with 80 mL of the proposed treatments. Four treatments were utilised: aqueous control; oil control; aqueous formulation and oil formulation with M. anisopliae s.l. (2.0 × 108 conidia/mL) (with 10 vessels per group). Pathogenicity against R. microplus was evaluated daily in order to identify the mortality of females and the recovery of larvae 42 days after the treatments began. Additionally, the fungus was re-isolated from deceased females and from the soil in the vessels. To evaluate the fungal viability in the soil, samples were collected before spraying and at the 3rd, 7th, 21st and 52nd days after treatments began. Oil formulation of M. anisopliae s.l. reduced the number of infesting larvae and promoted a percentage of control between 58.4% and 61.3%. Aqueous formulation promoted a percentage of control between 39.6% and 43.8%. In both groups, the persistence of the fungus in the soil was of up to 52 days. On the other hand, the control groups did not show any fungal growth. The fungal isolate, when formulated, has potential to be used in integrated control programmes against R. microplus. In addition, understanding the period of persistence of the fungus in the soil is important for the definition of protocols for fungal application in the field.