Soybean [Glycine max (L.) Merr] is the legume with the largest cultivated area worldwide and its yield depends largely on symbiotic nitrogen fixation and root architecture. This study aimed to explore the genetic variability of root architectural traits and di-nitrogen fixing activity in a small collection of nine European cultivars belonging to the same maturity group during their early stages. Cultivars were grown in Rhizotubes in the 4PMI high-throughput phenotyping platform and allowed us to capture images of the whole nodulated root system over time. New image analysis approaches were implemented to characterize root architecture and nodulation at high throughput. Significant genetic variability was identified for the width of the root system, the root density, and for the number of lateral roots. Differences in symbiotic fixation among the different genotypes were observed, while inoculating the same N2-fixing bacterial strain. Differences were also observed in the location of the nodules on the roots, the number of nodules, the specific N2-fixing activity of the nodules or the time when the highest rate of nodulation was reached. This study allowed us to highlight trade-offs among root and nodule traits, and structural and functional traits. Some more productive genotypes were characterized by a good balance between efficiency in producing efficient nodules and maintaining a large number of roots at the early developmental stages, that could result in a higher uptake of nutrients and water in these genotypes. Finally, both the image analysis approach and the results could be used for breeding programs of soybean, that could explicitly consider the root system architecture, when the plant interacts in symbiosis with N2-fixing bacteria.