Biphasic materials, comprised of an ordered arrangement of two different material phases within a material, have the potential for a wide variety of applications including filtration, protective clothing and tissue engineering. This study reports for the first time, a process for engineering biphasic Janus-type polymeric nanofiber (BJPNF) networks via the centrifugal jet spinning technique. BJPNF alignment and fiber diameter was dependent on fabrication rotational speed as well as solution composition. The biphasic character of these BJPNFs, which was controlled via the rotational speed of fabrication, was confirmed at the individual nanofiber scale using energy dispersive X-ray spectroscopy, and at the bulk, macro-scale using attenuated total reflectance-Fourier transform infrared spectroscopy. Biphasic character was also demonstrated at the functional level via differing affinities on either side of the BJPNF for cell attachment. Our work thus presents a method for fabricating BJPNF scaffold networks where there might be a need for different properties on either side of a material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2455-2464, 2017.