Entomopathogenic nematode (EPN) is an environmentally friendly alternative biocontrol agent for crop protection. Mass production of EPN is important for efficiency of such biological control. This is the first report exhibiting use of an available low-cost substrate, coarse ground soybean residue, in semi-solid media for culturing an EPN, Steinernema sp. NR01. The semi-solid medium containing 20 ml of basal medium along with the blend of soybean residue and yolk saline solution in ratio 1:1 increased the total number of EPN around 7-fold (1.47 × 104 nematodes/ ml) relative to the basal medium (0.20 × 104 nematodes/ ml) within 10 days post inoculation (dpi), with a significant difference at alpha level 0.05. The highest reproductive performance of females was found when EPN was cultured in the soybean residue and yolk media in 3:1 combination (0.50 × 104 nematodes/ ml), followed by 1:1 (0.49 × 104 nematodes/ ml) and 1:3 (0.42 × 104 nematodes/ ml) at 20 dpi. Moreover, in vitro cultured EPN could infect the wax moth (Galleria mellonella) and cause mortality within 48 hr post inoculation. Symbiotic bacteria were re-isolated from the infected wax moths. Thus, the combination of soybean residue and egg yolk in culture medium has improved the mass production of effective EPN and could be beneficial, adding value to soybean residue, and solving environmental problems from soymilk production waste.