The thermal and mechanical properties of two types of polyester nanofiber, poly (1,4-cyclohexanedimethylene isosorbide terephthalate) (PICT) copolymers and the terpolyester of isosorbide, ethylene glycol, 1,4-cyclohexane dimethanol, and terephthalic acid (PEICT), were investigated. This is the first attempt to fabricate PICT nanofiber via the electrospinning method; comparison with PEICT nanofiber could give greater understanding of eco-friendly nanofibers containing biomass monomers. The nanofibers fabricated from each polymer show similar smooth and thin-and-long morphologies. On the other hand, the polymers exhibited significantly different mechanical and thermal properties; in particular, a higher tensile strength was observed for PICT nanofiber mat than for that of PEICT. We hypothesized that PICT has more trans-configuration than PEICT, resulting in enhancement of its tensile strength, and demonstrated this by Fourier transform infrared spectroscopy. In addition, PICT nanofibers showed clear crystallization behavior upon increased temperature, while PEICT nanofibers showed completely amorphous structure. Both nanofibers have better tensile properties and thermal stability than the typical polyester polymer, implying that they can be utilized in various industrial applications.