Prolonged use of broad-spectrum antibiotics has led to the emergence of drug-resistant pathogens, both in medicine and in agriculture. New threats such as biological warfare have increased the need for novel and efficacious antimicrobial agents. Natural habitats not previously examined as sources of novel antibiotic-producing microorganisms still exist. One such habitat is the rhizosphere of desert shrubs. Here, we show that one desert shrub habitat, the rhizosphere of desert big sagebrush ( Artemisia tridentata) is a source of actinomycetes capable of producing an extensive array of antifungal metabolites. Culturable microbial populations from both the sagebrush rhizosphere and nearby bulk soils from three different sites were enumerated and compared, using traditional plate-count techniques and antibiotic activity bioassays. There were no statistical differences between the relative numbers of culturable non-actinomycete eubacteria, actinomycetes and fungi in the rhizosphere versus bulk soils, but PCR amplification of the 16S rRNA gene sequences of the total soil DNA and denaturing gradient gel electrophoresis showed that the community structure was different between the rhizosphere and the bulk soils. A high percentage of actinomycetes produced antimicrobials; and the percentage of active producers was significantly higher among the rhizosphere isolates, as compared with the bulk soil isolates. Also, the rhizosphere strains were more active in the production of antifungal compounds than antibacterial compounds. 16S rRNA gene sequence analysis showed that sagebrush rhizospheres contained a variety of Streptomyces species possessing broad spectrum antifungal activity. Scanning electron microscopy studies of sagebrush root colonization by one of the novel sagebrush rhizosphere isolates, Streptomyces sp. strain RG, showed that it aggressively colonized young sagebrush roots, whereas another plant rhizosphere-colonizing strain, S. lydicus WYEC108, not originally isolated from sagebrush, was a poor colonizer of the roots of this plant, as were two other Streptomyces isolates from forest soil. These results support the hypothesis that the rhizosphere of desert big sagebrush is a promising source of habitat-adapted actinomycetes, producing antifungal antibiotics.