Entomopathogenic nematodes are potential control agents for insect pests in cryptic habitats and are found in many environments worldwide. Native entomopathogenic nematodes were collected from March to June 2012 in soil from agave (Agave angustifolia Haw) fields in the Central Valleys of Oaxaca to evaluate potential for control of agave weevil, Scyphophorus acupunctatus Gyllenhal (Coleoptera: Curculionidae). The technique of insect-baiting with larvae of Galleria mellonella (L.) was used. Using sequencing internal transcribed spacer regions of ribosomal DNA and the 28s or subunit, Steinernema websteri Cutler & Stock and Steinernema carpocapsae (Weiser) Wouts, Mracek, Gerdin & Bedding nematodes were identified. The entomopathogenicity of the nematodes was verified by Koch's postulates. The efficacy of the strains at concentrations of 100, 500, and 1,000 infective juveniles per milliliter was tested on S. acupunctatus larvae in Petri dishes. Both nematode species at concentrations of 500 and 1,000 infective juveniles per larva killed 85–100% of agave weevils. The lethal mean concentrations (LC₅₀) were 168 infective juveniles of S. websteri and 147 of S. carpocapsae, while the LC₉₀ values were 890 and 3,739 infective juveniles per larva, respectively. This study is the first report of a native S. websteri strain in Mexico, which is proposed as a potential biological control agent against S. acupunctatus.