Understanding the structural stability of nanomaterials such as nanowires is imperative for their practical applications. Here, using single-shot ultrafast transmission electron microscopy, we captured the Plateau–Rayleigh instability behavior of silver nanowires decomposing into silver nanoparticles upon laser irradiation on nanosecond time scales. We found that this dynamic process can be accelerated by a factor of 10 through increasing the peak laser fluence from 6.03 to 14.60 mJ/cm². Moreover, by comparing the laser fluence required to initiate the Plateau–Rayleigh instability in silver nanowires on carbon and Formvar membranes, it was found that the main driving force for the Plateau–Rayleigh instability in this study was substrate heating. Finite element analysis and molecular dynamics simulations are also applied to estimate the temperature jump of the nanowires and atomistic behavior, respectively. In addition, the complex motions of silver nanowires under laser irradiation were systematically investigated by combining the results of ultrafast transmission electron microscopy with scanning electron microscopy characterizations and were determined to be related to nanowire–membrane interactions or pre-existing stresses on the nanowires, which in turn demonstrated the potential of ultrafast transmission electron microscopy for the characterization of nanomaterials and devices under extreme conditions.