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Pest control in Saudi Arabia is entirely relied on the application of chemical agents. Little information is known about the natural presence of Bacillus thuringiensis species that possess insecticidal activity in the environment of Saudi Arabia. It would be of interest to search for native species of toxic Bt strains that can be used in pest control management. Thus the aim of this study was to investigate the natural presence of Bacillus thuringiensis species that are toxic to pests in the environment of Makkah Province, western Saudi Arabia. A total of 100 soil samples and five dead larvae of Spodoptera littoralis (Lepidoptera) were examined for the presence of Bacillus thuringiensis. The bacterium was isolated by acetate-selective enrichment and plating. Identification of isolates was performed by microscopic examination, analysis of parasporal inclusions protein profiles by SDS-PAGE, toxicity assay, analysis of 16S rDNA genes and DNA sequencing for PCR products. The confirmed Bacillus thuringiensis isolates, eight in total, were recovered from 5% of soil samples and from 60% of dead larvae. These isolates exhibited strong activity against 1(st) instar larvae of S. littoralis. Although Bacillus thuringiensis was not found to be abundant in soil habitats in Makkah Province, the results suggest that the bacterium is part of the indigenous microflora of the area we have explored. This is the first report of the natural presence of lepidopteran-toxic strains of Bacillus thuringiensis in the environment of western Saudi Arabia, particularly in Makkah Province.