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The literature suggests that nitrogen (N) fertilization increases yield in soybean. This study aimed to investigate the effects of N fertilization on: (i) The performance of soybean, and (ii) the weed flora. A two-year field experiment was carried out in Agrinio, Western Greece. The experiment was set up in a randomized complete block design, with four organic fertilizer treatments and six replications. The four treatments included 0 kg N ha⁻¹ (N0/unfertilized control) and the application of 80 kg N ha⁻¹, 100 kg N ha⁻¹, and 120 kg N ha⁻¹. The application of 120 N kg ha⁻¹ resulted in the most notable increment of plant height (22.6–24%), biomass (10–13%), LAI values (14–17%), and yield (10–12%) compared to the N0. Compared to the N0, total weed biomass was increased by 26–32%, 34–49%, and 55–57% in N80, N100, and N120, respectively. The values of the H (Shannon), Dmg (Margalef), and J (Pielou) indices were unaffected by the fertilization, hence they did not affect weed biodiversity. CRI (crop resistance index), on the contrary, was negatively affected by N fertilization and was significantly reduced. Overall, our results indicate that the application of 80 kg N ha⁻¹ is more efficient, can effectively improve the soybean performance, and enhance its yield.