The use of chemical marker analysis for characterising microbial communities in organic dust samples is exemplified in a comparative study of dusts collected in a home and a swine confinement building, respectively. The chemical markers studied included 3-hydroxy fatty acids (markers of endotoxin), ergosterol (marker of fungal biomass), and muramic acid (marker of peptidoglycan/bacterial biomass). Samples were hydrolysed and subjected to various chemical manipulations for rendering the markers suitable for gas chromatography-tandem mass spectrometry analysis. Considerable differences between the dust samples were revealed. Swine dust contained 46 ng/mg of ergosterol (house dust 2.1 ng/mg), 0.096 nmol/mg of endotoxin (house dust 0.020 nmol/mg), and 483 ng/mg of muramic acid (house dust 366 ng/mg). The 3-hydroxy fatty acid and muramic acid results demonstrated a much higher proportion of Gram-negative bacteria to Gram-positives in swine dust than in house dust, and ergosterol results demonstrated a much higher proportion of fungi. The different distribution of 3-hydroxy fatty acids in the 2 samples illustrated differences in their flora of Gram-negative bacteria. The described method allows accurate determination of markers even when present down to trace levels in chemically complex matrices and should be useful in evaluating the role of microorganisms in the development of occupational lung disease, e.g. in agricultural environments.