Aims: Subterranean termites, Globitermes sulphureus is one of the significant pests for agricultural crops such as coconut and oil palm, and occasionally attacks building structure in Malaysia. Efforts to control subterranean termite infestations depended heavily on liquid termiticide applications. Natural pathogen of termites such as entomopathogenic fungi (EPF) is a promising alternative to chemical control. The objective of this study was to determine the most virulent EPF such as Metarhizium anisopliae, Beauveria bassiana and Paecilomyces sp. against the subterranean termites, G. sulphureus for the development of bio-insecticide for future use of termite control. Methodology and results: Subterranean termites were collected from mound-building termites found in Universiti Malaysia Terengganu (UMT). Cultivating, harvesting and counting of conidia of EPF were carried out to prepare the desired concentrations for screening test and bioassays, which were 1×103 , 1x105 , 1×107 and control. The pathogenicity testing was observed daily within 1 week under laboratory conditions. Screening showed that M. anisopliae was found to be the most virulent compared to B. bassiana and Paecilomyces sp., achieving 100% mortality within 3-4 days. M. anisopliae was then further tested on termites and it was found that concentration of 1×107 showed the lowest LT50 value, while LC50 of M. anisopliae in 1 day was 2.0151×106 . Conclusion, significance and impact of study: It could be concluded that M. anisopliae is the most virulent EPF against termites and the most effective concentration was ±107 followed by strains of B. bassiana and Paecilomyces sp. However, there is limited field evaluation of EPF against termites in Malaysia. Evaluation on the efficiency of M. anisopliae in the field should be conducted so that its efficacy could be proven and marketed.