Tissue engineering is a multidisciplinary field that has evolved in various dimensions in recent years. One of the main aspects in this field is the proper adjustment and final compatibility of implants at the target site of surgery. For this purpose, it is desired to have the materials fabricated at the nanometer scale, since these dimensions will ultimately accelerate the fixation of implants at the cellular level. In this study, electrospun polyurethane nanofibers and their analogous nanofibers containing MWCNTs are introduced for tissue engineering applications. Since MWCNTs agglomerate to form bundles, a high intensity sonication procedure was used to disperse them, followed by electrospinning the polymer solutions that contained these previously dispersed MWCNTs. Characterization of the produced nanofibers has confirmed production of different non-woven mats, which include random, semi-aligned and mostly aligned patterns. A simultaneous and comparative study was conducted on the nanofibers with respect to their thermal stability, mechanical properties and biocompatibility. Results indicate that the mostly aligned nanofibers pattern presents higher thermal stability, mechanical properties, and biocompatibility. Furthermore, incorporation of MWCNTs among the different arrangements significantly improved the mechanical properties and cell alignment along the nanofibers.