The aim of this work is to develop a series of advanced biobased tougheners for thermosetting epoxy resins suitable for high-performance applications. These bio-rubber (BR) tougheners were prepared via a one-step chemical modification of epoxidized soybean oil using biobased hexanoic anhydride. To investigate their toughening performance, these BR tougheners were blended with diglycidyl ether of bisphenol A epoxy monomers at various weight fractions and cured with anhydride hardeners. Significant improvements in fracture toughness properties, as well as minimal reductions in glass transition temperature (Tg), were observed. When 20 wt % of a BR toughener was utilized, the critical stress intensity factor and critical strain energy release rate of a thermosetting matrix were enhanced by >200 and >500%, respectively, whereas the Tg was reduced by only 20 °C. The phase-separated domains were evenly dispersed across the fracture surfaces as observed through scanning electron microscopy and atomic force microscopy. Moreover, domain sizes were demonstrated to be tunable within the micrometer range by altering the toughener molecular structure and weight fractions. These BR tougheners demonstrate the possibility of achieving toughness while having the thermal properties of standard bisphenol epoxy thermosetting resins.