We present a multifrequency radio investigation of the Crab-like pulsar wind nebula (PWN) G54.1+0.3 using the Very Large Array. The high resolution of the observations reveals that G54.1+0.3 has a complex radio structure which includes filamentary and loop-like structures that are magnetized, a diffuse extent similar to the associated diffuse X-ray emission. But the radio and X-ray structures in the central region differ strikingly, indicating that they trace very different forms of particle injection from the pulsar and/or particle acceleration in the nebula. No spectral index gradient is detected in the radio emission across the PWN, whereas the X-ray emission softens outward in the nebula. The extensive radio polarization allows us to image in detail the intrinsic magnetic field, which is well-ordered and reveals that a number of loop-like filaments are strongly magnetized. In addition, we determine that there are both radial and toroidal components to the magnetic field structure of the pulsar wind nebula. Strong mid-IR emission detected in Spitzer Space Telescope data is closely correlated with the radio emission arising from the southern edge of G54.1+0.3. In particular, the distributions of radio and X-ray emission compared with the mid-IR emission suggest that the PWN may be interacting with this interstellar cloud. This may be the first PWN where we are directly detecting its interplay with an interstellar cloud that has survived the impact of the supernova explosion associated with the pulsar's progenitor.