Soil-borne fungi are considered as major pathogens to many plants and can cause a severe economic damage. Early detection and identification of these pathogens is very important and might be critical for their control. The available methods for identification of fungi like molecular biology, serological tests and PCRs tests (polymerase chain reaction) are time consuming and not always very specific. Fourier-transform infrared (FTIR) attenuated total reflection (ATR) spectroscopy, is considered to be a comprehensive and sensitive method for detection of molecular changes in intact cells.In the present study we used FTIR–ATR as a sensitive and effective assay for the detection and discrimination between different fungal genera. Our results showed significant spectral differences between the various examined fungi genera. These results proved the possibility of discrimination between these fungi on the genus level.