When a sessile droplet containing a solute in a volatile solvent evaporates, flow in the droplet can transport and assemble solute particles into complex patterns. Transport in evaporating sessile droplets has largely been examined in solvents that undergo complete evaporation. Here, we demonstrate that flow in evaporating aqueous sessile droplets containing type I collagen-a self-assembling polymer-can be harnessed to engineer hydrated networks of aligned collagen fibers. We find that Marangoni flows direct collagen fiber assembly over millimeter-scale areas in a manner that depends on the rate of self-assembly, the relative humidity of the surrounding environment, and the geometry of the droplet. Skeletal muscle cells that are incorporated into and cultured within these evaporating droplets collectively orient and subsequently differentiate into myotubes in response to aligned networks of collagen. Our findings demonstrate a simple, tunable, and high-throughput approach to engineer aligned fibrillar hydrogels and cell-laden biomimetic materials.