| Summary: | <i>Rhipicephalus microplus</i> is a persistent ectoparasite of cattle that causes bovine anaplasmosis and babesiosis, causing economic losses worldwide. Chemical treatment is the primary method for tick control, but the emergence of pesticide-resistant ticks is a major challenge. Alternative biocontrol strategies utilizing entomopathogenic microorganisms are being explored. This study aimed to validate the species identification and assess the efficacy of four strains of <i>Staphylococcus</i> bacteria (<i>S. shinii</i> S1 and S-2, <i>S. succinus</i>, and <i>S. xylosus</i>) previously reported as being entomopathogenic to <i>R. microplus</i> ticks. According to the bioassays, <i>S. shinii</i> S-1 exhibited the greatest degree of reproductive inhibition (47%), followed by <i>S. succinus</i> (44.3%) at a concentration of 1 × 10<sup>8</sup> cfu/mL. <i>S. xylosus</i> displayed decreased reproductive inhibition (6.3%). In an additional bioassay, <i>S. shinii</i> S-1 exhibited a significant larval mortality of 67.63%, followed by <i>S. succinus</i> with 66.75%, <i>S. shinni</i> S-2 with 64.61%, and <i>S. xylosus</i> with 28.18% mortality. The common signs of infection observed on these ticks included swelling, yellowish exudate on the hypostome, and reduced limb mobility and color change, except for <i>S. succinus</i>, which did not cause color changes. These bacteria were naturally found on bovine skin. However, further studies are needed to confirm their potential as promising alternatives or complementary agents to existing acaricidal compounds.
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