Recent evidence indicates that both cardiovascular fitness and gross motor skill performance are related to enhanced neurocognitive functioning in children by influencing brain structure and functioning. This study investigates the role of white matter microstructure in the relationship of both cardiovascular fitness and gross motor skills with neurocognitive functioning in healthy children. In total 92 children (mean age 9.1 years, range 8.0-10.7) were included in this study. Cardiovascular fitness and gross motor skill performance were assessed using performance-based tests. Neurocognitive functioning was assessed using computerized tests (working memory, inhibition, interference control, information processing, and attention). Diffusion tensor imaging was used in combination with tract-based spatial statistics to assess white matter microstructure as defined by fractional anisotropy (FA), axial and radial diffusivity (AD, RD). The results revealed positive associations of both cardiovascular fitness and gross motor skills with neurocognitive functioning. Information processing and motor response inhibition were associated with FA in a cluster located in the corpus callosum. Within this cluster, higher cardiovascular fitness and better gross motor skills were both associated with greater FA, greater AD, and lower RD. No mediating role was found for FA in the relationship of both cardiovascular fitness and gross motor skills with neurocognitive functioning. The results indicate that cardiovascular fitness and gross motor skills are related to neurocognitive functioning as well as white matter microstructure in children. However, this study provides no evidence for a mediating role of white matter microstructure in these relationships.