The purpose of this study was to quantify the impact of effective nodulation on yield and quality of organically produced soybean and to assess three methods for the quantification of symbiotic N2 fixation. We used (i) the natural abundance (NA) method which relies on isotopic differences in shoot N between the N-fixing legume and non-fixing reference plants, (ii) a method independent of reference plants that is based on within-soybean N isotopic fractionation and (iii) the xylem solute method, in which the proportion of N transported into shoot material in the form of ureides that are specific to N-fixing soybeans is quantified. Effectively nodulated soybeans produced greater yields of better quality than non-nodulated soybeans in three separate experiments conducted on fertile, organically managed fields. Employing the NA method with non-nodulated soybeans as reference plants, symbiotic N2 fixation contributed 40–52% to overall N uptake by nodulated soybeans. These estimates for the percentage of soybean N derived from atmosphere (%Ndfa) were significantly increased when isotopic data from naturally occurring weeds were used in the calculation. Xylem ureide concentrations were not correlated with %Ndfa determined by the NA method. NA-determined %Ndfa were closely correlated with differences in the isotopic composition between soybean shoot N and root including nodule N (Δδ15N = δ15N shoot − δ15N root). Further studies are required to assess the potential suitability of Δδ15N to quantify soybean %Ndfa independent of reference plants.