The objectives of the present study were to isolate Beauveria bassiana strains from cattle farm soils, analyze the phylogenetic relationships among the fungal strains isolated from these soils, and determine the acaricidal effect of B. bassiana isolates on engorged Rhipicephalus microplus tick strains resistant or susceptible to chemical acaricides. Six strains of B. bassiana were obtained and isolated from cattle farm soils in the Mexican tropics using the Galleria bait method, and their acaricidal effect was assessed against 2 populations of R. microplus (“Media Joya” chemical acaricide-resistant strain or “CLAR” chemical acaricide-susceptible strain) using the adult immersion test. The BbV03 strain produced 86.7% and 60% mortality in resistant and susceptible ticks on day 20, respectively, whereas the BbV04 strain produced 66.7% and 53.5% mortality in resistant and susceptible ticks on day 20, respectively. The BbV03 and BbV04 strains reduced egg laying on both R. microplus populations. There was no statistical difference in the acaricidal effect of B. bassiana strains among chemical acaricide-susceptible or -resistant R. microplus populations (P > 0.05). The BbV03 strain was the most virulent against R. microplus with an LC50 of 2 × 107 and LC99 of 7 × 108 conidia/ml. We found that the 6 B. bassiana isolated clustered in the same clade with other previously reported B. bassiana strains (from GenBank) but were separated into 3 different sub-clades. This study shows that some B. bassiana strains are a promising coadjuvant alternative for biological tick control, including tick populations that are resistant to chemical acaricides. Beauveria bassiana is present in the pastures of tropic cattle farms, and there are genetic variations between B. bassiana strains living in this ecosystem that might play an important role in the natural control of R. microplus in cattle farm paddocks.