Bacillus thuringiensis(Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select newBtcandidates challenging the emergence of insect's resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae,Grapholita molesta, andSpodoptera exigua) were conducted using spore-crystal mixtures and supernatant cultures of 49Btisolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of "functional mortality" was used to discriminate between "nontoxic" and "toxic" isolates. The toxicity of manyBtisolates competed with that ofBtk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore-crystal mixtures and supernatant cultures against the three lepidopteran species. TheBtisolates Bl4NA and Bl9NA express a protein of 130 kDa whereas theBtisolate Bl5NA expresses a protein of 65-70 kDa. The LC-MS/MS results indicate that the major peptides in the 130 kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70 kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison toBtk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likelyVip3Aa inBl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these threeBtstrains do not produce beta-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.