Lysobacter enzymogenes C3 is a predatory strain of Gram-negative gliding bacteria that produces antifungal antibiotics by the polyketide synthetic pathway. Outer membrane vesicles (OMV) are formed as a stress response and can deliver virulence factors to host cells. The production of OMV by C3 and their role in antifungal activity are reported here. Vesicles in the range of 130 to 150 nm in diameter were discovered in the cell-free supernatants of C3 cultures. These OMV contain molecules characteristic of bacterial outer membranes, such as lipopolysaccharide and phospholipids. In addition, they contain chitinase activity and essentially all of the heat-stable antifungal activity in cell supernatants. We show here that C3 OMV can directly inhibit growth of the yeast Saccharomyces cerevisiae as well as that of the filamentous fungus Fusarium subglutinans. The activity is dependent on physical contact between OMV and the cells. Furthermore, fluorescent lipid labeling of C3 OMV demonstrated transfer of the membrane-associated probe to yeast cells, suggesting the existence of a mechanism of delivery for membrane-associated molecules. Mass spectrometric analysis of C3 OMV extracts indicates the presence of molecules with molecular weights identical to some of the previously identified antifungal products of C3. These data together suggest that OMV act as an important remote mobile component of predation by Lysobacter.