We report on direct force measurements between single melt-electrospun 1,3,5-cyclohexanetrisamide (CTA) fibers in crossed-cylinder geometry. The two CTA compounds selected for this study differ in their peripheral substituents: aliphatic (-C₁₀H₂₁) and fluorinated chains (-CH₂C₆F₁₃), respectively. Melt-electrospinning of the CTAs results in smooth and circular fibers with diameters of about 5 μm. Individual segments of these fibers were attached to tipless atomic force microscope (AFM) cantilevers and used to measure long-range interaction forces versus a second fiber from the same compound in crossed-cylinder geometry. This geometry is well-known from the surface force apparatus and allows for the normalization of forces according to the Derjaguin approximation. From symmetrical measurements, i.e. measurements between fibers from the same type of CTA, we quantify the diffuse layer properties in aqueous electrolyte solutions within the framework of the Derjaguin, Landau, Verwey and Overbeek (DLVO) theory. Apparent diffuse layer potentials resulting from the fits to the full solutions of the Poisson–Boltzmann equation show that the fiber surfaces bear a negative surface charge. Most likely, the origin of charging is the adsorption of hydroxyl ions as residual charges from the electrospining process would be compensated upon the immersion in the electrolyte solutions. Such ion adsorption processes are well-known for other hydrophobic surfaces such as aliphatic and fluorinated self-assembled monolayers (SAMs) (terminating with [sbnd]CH₃ and [sbnd]CF₃ groups). The apparent diffuse layer potentials for CTA fibers are comparable to the values reported for these SAMs.