Datura, a medicinal plant, has been used for centuries as a remedy for human diseases because of its richness in alkaloids of therapeutic values. Besides alkaloids, studies on other bioactive substances in Datura such as phenylpropanoids, flavonoids or amino acid are less advanced. However, the study of these compounds can offer new perspectives in biological, medicinal and pharmaceutical research. In addition, the application of Plant Growth Promoting Rhizobacteria (PGPR) can influence the concentrations of certain plant metabolites such as amino acids, and consequently, the production of these substances can be enhanced. Analyses of amino acids and their derivatives by Gas chromatography-Electron impact/Time of flight-Mass spectrometry (GC-EI/TOF-MS) revealed variations in roots or in leaves of Datura stramonium. The following amino acids and their derivatives were present in this species: alanine, alanine 3-cyano-, alanine beta-, arginine, asparagine, aspartic acid, butanoic acid 4-amino-, butyro-1,4-lactam, glutamic acid, glutamine, glycine, histidine, homocysteine, homoserine, isoleucine, leucine, lysine, ornithine, ornithine-1,5-lactam, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine and valine. Application of PGPR to Datura stramonium affected significantly the levels of some amino acids either by increasing or by reducing their content mainly in the leaves and in some cases also in the roots.