Background The prevalence of allergic diseases has increased particularly over the past 30-40 years. A reduced microbial stimulation during infancy may result in a development of a disturbed balance between Th1- and Th2-like immunity. The gut flora is, quantitatively, the most important source for such stimulation. Objective The aim of the study was to compare the gut microbial flora in 25 allergic and 47 nonallergic 13-month-old infants (range 11-18), through analysing microflora-associated biochemical markers in faeces. Methods Microflora associated characteristics (MACs) were assessed by determining the concentrations of eight different short chain fatty acids and the conversion of cholesterol to coprostanol by gas chromatography. Faecal tryptic activity was analysed spectrophotometrically. Results The allergic infants had lower levels of propionic, i-butyric, butyric, i-valeric and valeric acid. In contrast, they had higher levels of the rarely detected i-caproic acid, which has been associated with the presence of Clostridium difficile. Furthermore, the allergic infants had higher relative distribution of acetic and i-caproic acid. None of the other parameters differed between the groups. Conclusion The results demonstrate differences in the MACs between allergic and nonallergic infants, indicating differences in the composition of the gut flora. that may disturb the development of a normal Th1-/Th2-balance in allergic children.