This paper reports single-molecule tracking (SMT) measurements of the diffusion behaviors of individual, anionic sulforhodamine B (SRB) dye molecules in a series of poly(ethylene oxide) (PEO) films, aimed at clarifying the influences of the molecular weight, network plasticization, and thermal annealing on such dynamics. Micrometer-thick PEO films were prepared by drop-casting from its aqueous (0.2%, 1 nM SRB) solution, followed by drying in air and thermal annealing at 90 °C for 36 h. The diffusion of individual SRB occurring within the amorphous domains was recorded at different relative humidities (5-95%) to characterize the microscale domains' local aspect-ratio, orientation, and molecular permeability at high spatial resolution. The results revealed the involvement of crystalline phases in confining SRB diffusion to submicron distances and guiding longer-range diffusion along one-dimensional-like amorphous morphologies. Upon annealing, amorphous domains were wider, more continuous, and more permeable to SRB probes. The enhanced transport in plasticized PEO, as reflected by the higher SRB mobility and diffusivity, was linked to the polymer's higher chain and segmental mobilities and reduced hydrogen-bonding interactions. This work has demonstrated the usefulness of SMT for an advanced characterization of solid polymer electrolytic films, highly beneficial for the development of safer lithium-ion batteries.