The livestock industry significantly impacts Malaysia’s agriculture, with cattle being a key source of meat and milk. However, cattle are vulnerable to tick-borne pathogens like Anaplasma and Babesia, infecting over 80% of the cattle population, thus threatening animal health and the country’s goal of meat self-sufficiency. The primary vector, Rhipicephalus (Boophilus) microplus, poses a serious economic burden on the industry. Traditionally, chemical acaricides have been used for tick control, but their overuse has resulted in resistance, environmental contamination, and health risks. Therefore, there is growing interest in exploring alternative approaches, such as entomopathogenic fungi. To address these challenges, this study evaluated the effectiveness of Metarhizium anisopliae isolated from Malaysia against R. microplus using the Adult Immersion Test. Engorged female ticks were exposed to M. anisopliae isolates at a concentration of 108, with mortality rates tracked over 14 days. Results showed pathogenicity across all isolates, with mortality rates ranging from 36.67 to 83.33%. The PR1 isolate had the highest virulence and shortest LT50 of 10 days. A moderate negative correlation was found between tick size and mortality. These findings emphasize the influence of tick size on tick mortality. Although no isolate achieved 100% mortality, PR1 was notably effective, killing the highest percentage of ticks quickly and significantly reducing egg production compared to the control and other isolates. Overall, this study underscores the potential of Malaysia-isolated M. anisopliae in the management of adult R. microplus, offering insights into alternative strategies for pest control in the livestock sector.