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The larva of Scrobipalpuloides absoluta, a South American moth, is the most devastating insect pest of tomato production in Chile. The potential for using bacterial insecticides was studied analysing the relative toxicity of native Bacillus thuringiensis (BT) isolates belonging to the Chilean collection. The polymerase chain reaction (PCR) technique was used in order to facilitate the prescreening. Mixtures of homologous specific primers to regions within genes encoding CryI, CryIII and CryIV crystal proteins were employed to generate a PCR product profile of each BT isolate. Four isolates were selected and further characterized by means of SDS-PAGE, Western blot and bioassays on fourth-instar S. absoluta larvae. Relative toxicities were evaluated by LD50 determinations. The entomocidal activity of isolate 121e, an autoagglutinating strain, was threefold higher than toxin synthesized by B. thuringiensis var. kurstaki. This native strain was also active against Culex pipiens larvae, although much less than towards S. absoluta.