Potential applications of atomic force microscopy (AFM) in textiles are explained. For this purpose samples were carefully selected from both natural and synthetic fibers. Cotton, wool, conventional polyethylene terepthalate (PET), antibacterial PET, and antistatic PET were investigated by means of 3D topography imaging, phase imaging, and calculation of their Rq values. The distribution of the additives in the cross sections of antibacterial PET and antistatic PET were analyzed. Moreover, differences between inner and outer cross section of trilobal PET was observed by force spectroscopy. The results are discussed considering the fiber properties. It is concluded that AFM is a powerful tool to investigate different properties of textile fibers, and it gives valuable information.