This is the first study to empirically evaluate the mortality of Amblyomma mixtum larvae caused by native entomopathogenic fungi (EPF) from cattle farm soils and the impact of the physicochemical conditions of those soils on the ability of EPF to kill ticks. The efficacy of 25 EPF strains isolated from cattle farms soils in Veracruz, Mexico, against A. mixtum ticks was evaluated using a larval immersion test. Physicochemical analyses of the soils where the EPF were isolated were carried out, and the results were correlated with the obtained mortality. The MaV25 strain showed a mortality of 36.55% (P < 0.05), followed by MaV57 with 27.30%, MaV08 with 26.21%, and MaV31 with 24.32% (P < 0.05). Nitrogen and potassium are nutritional components of soils that seem to be associated with mortality caused by the evaluated fungal strains. This is the first study in Mexico where the effect of EPF against A. mixtum is evaluated and also is the first study in the world that uses native strains from livestock soils against larvae of this tick. It is necessary to determine the virulence characteristics of EPF on A. mixtum to improve knowledge of fungus–tick interactions (Metarhizium anisopliae–A. mixtum).