Cortical activity during simple unimanual actions is typically lateralized to contralateral sensorimotor areas, while a more bilateral pattern is observed with an increase in task demands. In parallel, increasing task demands are associated with subtle mirror muscle activity in the resting hand, implying a relative loss in motor selectivity. The corpus callosum (CC) is crucially involved in unimanual tasks by mediating both facilitatory and inhibitory interactions between bilateral motor cortical systems, but its association with mirror motor activity is yet unknown. Here, we used diffusion-weighted imaging and bilateral electromyographic (EMG) measurements during a unimanual task to investigate potential relationships between white matter microstructure of the CC and mirror EMG activity. Participants performed an unimanual pinch force task with both hands alternatively. Four parametrically increasing force levels were exerted while EMG activity was recorded bilaterally from first dorsal interosseus muscles. Consistent with previous findings, mirror EMG activity increased as a function of force. Additionally, there was a significant relationship between the slope of increasing mirror EMG during right-hand contractions and fractional anisotropy in transcallosal fibers connecting both M1. No significant relationships were found for fibers connecting dorsal premotor cortices or supplementary motor area, indicating the local specificity of the observed brain–physiology relationship.