Rapid emergence of bacterial antibiotic resistance has diminished the effectiveness of nearly all clinical antibiotics; thus, the search for novel compounds must continue in order to replenish the antibiotic drug pipeline. Beyond the clinic, antibiotics and antibiotic-producing organisms are additionally important in biotechnology and biological control applications. Natural products are the most logical starting point for discovering new molecules with antibiotic activity, and natural rhizosphere environments remain a practical source of new antibiotic producers. Increased success of culturing natural soil bacteria, combined with the knowledge that for every teaspoon of productive soil there are approximately 100 million to 1 billion bacteria, highlight the need to explore and exploit natural rhizosphere communities. Here, a cultured-based study reports isolation of 25 antibiotic-producing bacteria were isolated from soil- and plant-associated environments. Each bacterium was phylogenetically identified via 16S rDNA amplification and sequencing, and the spectrum of antibiotic activity was characterized against a range of Gram-positive and Gram-negative bacterial targets. Antagonistic organisms include members of several common genera, including Pseudomonas, Serratia and Streptomyces, as well as less characterized groups including Janthinobacteria and Rahnella species. Antifungal activity against the pathogenic fungus Fusarium was also assessed, with 16% of the antibacterial isolates also exhibiting antifungal activity. Ongoing work seeks to characterize the bacterial producers and the natural products being produced, with the long-term goal of discovery of novel compounds with clinical and agricultural applications.