A Bacillus thuringiensis strain (MnD) was isolated from Malacosoma neustria L (Lasiocampidae, Lepidoptera) insect. This isolate was compared with the reference strains, by means of electron microscopy, SDS-PAGE analysis, plasmid pattern, cry gene content, and insecticidal activity. Isolate MnD produced bipyramidal and cubical-shaped parasporal inclusions. The presence of Cry1 and Cry2 proteins in this isolate was confirmed by observation of 130 and 65 kDa proteins by SDS-PAGE. Plasmid pattern of MnD showed similar bands to those of the reference strain, B. thuringiensis subsp. kurstaki HD-1. The only noticeable difference was the lack of two large plasmids (3.5 and 2.9 kbp). PCR analysis showed that the strain has two different cry genes, cry1 and cry2. Toxicity tests were performed against seven insect species from Lepidoptera; Coleoptera and Diptera groups. Crystal-spore suspensions showed toxicity only against species of Lepidoptera (Malacosoma neustria, Lymantria dispar and Hyphantria cunea). This is the first study showing the significance future of a B. thuringiensis isolate from M. neustria.