A method based on polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is proposed for determining the infrared dichroic absorption ratio of a single fiber from a sample deposited flat on a germanium crystal without the requirement of fiber orientation. The method shows its efficiency on cellulose fibers of paper and has been applied to protein fibers (type I collagen and β-amyloid) and polysaccharide fibers (cellulose and starch). The method gives access to the dichroic ratio of strong absorptions bands, which is not easily accessible with conventional absorption techniques. Then, the orientation of the molecular groups of organic fibers can be easily determined by polarized ATR-FTIR spectroscopy. By extension, this method will be useful to determine the molecular orientation of fibers in structured complex samples, such as biological tissues and plants. Spatially resolved information on the organization of the fiber network will be easily extracted by utilizing a focal plane array detector for imaging measurements.