The aim of this research was to assess the efficacy of Metarhizium anisopliae (Ma) and diatomaceous earth (De), both individually and in combination, for the control of Rhipicephalus microplus ticks under laboratory and field conditions. In vitro experiments involved testing four concentrations of Ma (25%, 50%, 75%, and 100%), four concentrations of De (25%, 50%, 75%, and 100%), and three combination treatments (Ma25De75, Ma50De50, Ma75De25), alongside a control treatment of sterile distilled water with 0.1% (v/v) Tween 80, to evaluate their impact on R. microplus larvae. Laboratory trial results demonstrated that the application of Ma at 25% and 50%, as well as De at all concentrations, and the three combination treatments, led to a larval mortality exceeding 97% over a seven-day period. In contrast, individual treatments with Ma100 and Ma75 resulted in approximately 50% larval mortality. Subsequent field evaluations focused on Ma25De75 and Ma50De50 treatments for adult tick control. The application of Ma25De75 and Ma50De50 exhibited progressive increases in efficacy, reaching 97% and 88%, respectively, 21 days post-application. After the second application, efficacy further improved to 100% and 94.8% for Ma25De75 and Ma50De50, respectively. In conclusion, Ma25 and Ma50, along with all concentrations of De, proved to be effective options for controlling R. microplus larvae. Furthermore, a potential synergistic effect between M. anisopliae conidia and De was observed, demonstrating high efficacy rates of 100% in vitro for larvae and 88% to 100% in the field for adult R. microplus tick control.