Early maturing varieties of soybean have a high yield potential in Europe, where the main biotic threat to soybean cultivation are root lesion nematodes (Pratylenchus spp.). Nitrogen fixation in root nodules by highly efficient inoculants of Bradyrhizobium japonicum is an incentive to grow soybean in low-input rotation systems. We investigated density-dependent effects of Pratylenchus penetrans on nitrogen fixation by co-inoculated B. japonicum. Less than 130 inoculated nematodes affected the number and weight of nodules, the density of viable bacteroids in nodules, and nitrogen fixation measured as concentration of ureides in leaves. With more inoculated nematodes, the percentage that invaded the roots increased, and adverse effects on the symbiosis accelerated, leading to non-functional nodules at 4,000 and more nematodes. When P. penetrans invaded roots that had fully established nodules, growth of nodules, density of bacteroids, and nitrogen fixation were affected but not the number of nodules. In contrast, nodulation of already infested roots resulted in a high number of small nodules with decreased densities of bacteroids and nitrogen fixation. P. penetrans invaded and damaged the nodules locally, but they also significantly affected the nodule symbiosis by a plant-mediated mechanism, as shown in an experiment with split-root systems.