Our objective was to identify, for diagnostic purposes, potential volatile biomarkers of human microbial pathogens. We analysed the head space of cultures of medically important bacterial and fungal respiratory pathogens for 2-Pentylfuran (2PF) production through the use of Solid Phase Micro Extraction (SPME) and Gas Chromatography/Mass Spectroscopy (GC/MS). Our results confirm that 2PF is consistently produced by Aspergillus fumigatus, Fusarium spp., Aspergillus terreus, Aspergillus flavus and to a lesser extent by Aspergillus niger. 2-Pentylfuran was not detected from most of the bacterial strains except for Streptococcus pneumoniae. In human studies, four litre breath samples were collected from patients with cystic fibrosis (CF), with and without colonisation by A. fumigatus and other pathogens, as well as healthy volunteers. 2-Pentylfuran was detected in breath samples collected from 4/4patients with CF and A. fumigatus colonization, 3/7patients with CF and no microbiological evidence of A. fumigatus and 0/10healthy control individuals. These results suggest that 2PF may be a biomarker for lung colonization/infection by fungal pathogens. To our knowledge, this is the first report describing the detection in breath samples of a volatile biomarker of a pathogen resident in the lungs. Breath analysis has the potential of being a non-invasive diagnostic method of detecting respiratory infection including invasive aspergillosis