A new actinomycete strain called TN97 was isolated from Tunisian oasis soil and selected for its antimicrobial activities against bacteria and fungi. Cultural characteristic studies strongly suggested that this strain belongs to the genus Streptomyces. Nucleotide sequence of the 16S rRNA gene (1508 pb) of the TN97 strain exhibited high similarity to the Streptomyces 16S rRNA genes. This similarity reaches 96% with the 16S rRNA gene of Streptomyces lipmanii. In liquid medium, maximal biological activity production was obtained when glycerol or fructose were added at 1% (w/v). Extraction of 10 1 fermentation broth of the Streptomyces sp. TN97 strain, grown in tryptic soy broth (TSB) medium supplemented with 1% (w/v) of glycerol, and various separation and purification steps, led to the isolation of four pure bioactive molecules. The chemical structure of these compounds was established on the basis on their IR, ESI-MS and EI-MS, H-1 and C-13/APT NMR data and by comparison with reference data from literature. These compounds which are produced simultaneously by the Streptomyces sp. TN97 belong to three different families: two diketopiperazine derivatives [cis-cyclo (Leucyl-Prolyl) and cis-cyclo (L-phenyl, L-prolyl)], an isocoumarin derivative (the 6,8-dihydroxy-3-methylisocoumarin) and the N-acetyl-tyramine. (c) 2006 Elsevier Ltd. All rights reserved.