A new actinomycete strain designated US24 producing antibacterial activities against Gram-positive and Gram-negative bacteria was isolated from Tunisian soil. Culture characteristic studies strongly suggested that the US24 strain belonged to the genus Streptomyces. Analysis of the nucleotide sequence of the 16S rRNA gene of the Streptomyces sp. US24 strain showed high similarity (98%) with the 16S rRNA gene of Streptomyces caelestis which produces two antibiotics, niddamycin and celesticetin. Study of the influence of different nutritional compounds on antibiotic biosynthesis showed that the highest antibacterial activities were obtained when starch at 1% (w/v) was used as sole carbon source in the presence of traces of mineral oligoelements. Application to the supernatant culture of the Streptomyces sp. US24 strain of various separation steps led to isolation of two pure active molecules having a retention time of 34 and 37.26 min, respectively. P(34 min) possessed antibacterial activity against Gram-positive and Gram-negative bacteria, whereas P(37.26 min) inhibited only Gram-positive bacteria. Partial characterization of the P(34 min) molecule using spectroscopic studies showed that this active molecule is different from the two antibiotics produced by the S. caelestis strain.