Henequen natural fiber-reinforced poly(butylene succinate) biocomposites were prepared through a resin microdroplet formation on a single fiber and also fabricated by a compression molding technique using chopped henequen fibers, surface-treated with electron beam irradiation (EBI) at various dosages. The effect of EBI treatment on the surface characteristics and dynamic mechanical properties of henequen fibers was investigated using SEM, XPS and DMA methods. respectively. Also, the interfacial behavior of biocomposites was explored through a single fiber microbonding test and fracture surface observations. The result indicates that the interfacial shear strength (EFSS) of biocomposites greatly depends on the EBI treatment level on the henequen fiber surface. This study also suggests that appropriate modification of natural fiber surfaces at an optimum EBI dosage significantly contributes to improving the interfacial properties of biocomposites.