Polymeric electrospun nanofibers have extensive applications in filtration, sensing, drug delivery, and tissue engineering that often require the fibers to be patterned or integrated with a larger device. Here, we describe a highly versatile in situ strategy for three-dimensional electrospun fiber patterning using collectors with an insulative surface layer and conductive recessed patterns. We show that two-layer collectors with pattern dimensions down to 100-micrometers are easily fabricated using available laboratory equipment. We use finite element method simulation and experimental validation to demonstrate that the fiber patterning strategy is effective for a variety of pattern dimensions and fiber materials. Finally, the potential for this strategy to enable new applications of electrospun fibers is demonstrated by incorporating electrospun fibers into dissolving microneedles for the first time. These studies provide a framework for the adaptation of this fiber patterning strategy to many different applications of electrospun fibers.