In this paper a method of electrospinning conducting and nonconducting biphasic Janus nanofibers using microfluidic polydimethylsiloxane (PDMS)-based manifolds is described. Key benefits of using microfluidic devices for nanofiber synthesis include rapid prototyping, ease of fabrication, and the ability to spin multiple Janus fibers in parallel through arrays of individual microchannels. Biphasic Janus nanofibers of polyvinylpyrrolidone (PVP)+polypyrrole (PPy)/PVP nanofibers with an average diameter of 250 nm were successfully fabricated using elastomeric microfluidic devices. Fiber characterization and confirmation of the Janus morphology was subsequently carried out using a combination of scanning electron microscopy, energy dispersion spectroscopy, and transmission electron microscopy.