The sweetpotato weevil, Cylas formicarius (Coleoptera: Brentidae), is a major pest affecting sweet potato production in the field and quality during storage. Its nocturnal, subterranean lifestyle complicates control with synthetic insecticides, and insufficient knowledge of safe application methods exacerbates health risks while promoting resistance development. Biological control employing entomopathogenic fungi, including Metarhizium anisopliae, provides an eco-friendly solution. The present research focused on isolating and characterizing M. anisopliae from infected adult C. formicarius specimens and assessing its biocontrol effectiveness against this pest. Infected weevils were collected from the National Pingtung University of Science and Technology, Taiwan. The isolated fungus was identified as M. anisopliae. The effects of temperature variations and culture media on fungus revealed 25±1 °C as the optimal temperature, with the Sabouraud Dextrose Agar (SDA) medium surpassing both Potato Dextrose Agar (PDA) and Maltose Extract Agar (MEA) in spore production. The pathogenic effects of M. anisopliae on C. formicarius were analyzed using scanning electron microscopy to examine the fungal infection process. Adult mortality rates were 96.67, 93.33, and 50% at conidia concentrations of 1.2 × 10 8, 1 × 10 7, and 6.2 × 10 6 conidia/mL, respectively, seven days after treatment (DAT). The median lethal concentration 50 (LC 50) and LC 90 of M. anisopliae Pm04 against C. formicarius was calculated as 0.16 × 10 8 and 0.59×10 8 conidia/mL respectively. These findings indicate that the native M. anisopliae strain Pm04 has high virulence against C. formicarius, highlighting its promise as a viable biocontrol agent for managing field pests.