A novel strain of fluorescent pseudomonad (PB-St2) was isolated from surface-sterilized stems of sugarcane grown in Pakistan. The bacterium was identified as Pseudomonas aurantiaca on the basis of 16S rRNA gene sequence analysis and results from physiological and biochemical characteristics carried out with API50 CH and QTS 24 bacterial identification kits. Assays using substrate-specific media for enzymes revealed lipase and protease activities but cellulase, chitinase, or pectinase were not detected. The bacterium was unable to solubilize phosphate or produce indole acetic acid. However, it did produce HCN, siderophores, and homoserine lactones. In dual culture assays on agar, the bacterium showed antifungal activity against an important pathogen of sugarcane in Pakistan, namely Colletotrichum falcatum, as well as for pathogenic isolates of Fusarium oxysporium and F. lateritium but not against F. solani. The antifungal metabolites were identified using thin-layer chromatography, UV spectra, and MALDI-TOFF spectra and shown to be phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine (2-OH-PHZ), and N-hexanoyl homoserine lactone (HHL) (assessed using only TLC data). The capacity of this bacterium to produce HCN and 2-OH-PHZ, as well as to inhibit the growth of C. falcatum, has not been previously reported.