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ABSTRACT The application of flumioxazin and imazethapyr can affect the interactions between rhizobia and soybean in the rhizosphere. However, it remains unclear the effect of both herbicides, even when applied in mixture, on early nodulation and biological N fixation (BNF) in soybean, mainly related to some important biochemical traits in nodules. This study assessed the effect of flumioxazin, imazethapyr and their mixture on early nodulation and BNF in rhizosphere of soybean. The plant emergence, early nodulation, growth, and N accumulation were evaluated in soybean grown in sandy and clayey soils. The leghemoglobin content and glutamine synthetase (GS) activity were also assessed in fresh nodules. Individually, flumioxazin and imazethapyr did not affect the plant emergence, nodulation and N accumulation in soybean, while the mixture decreased significantly these parameters. Also, the values of leghemoglobin content and GS activity did not decrease with the application of the herbicides individually. The application of herbicides imazethapyr and flumioxazin separately negatively influenced the growth of soybean, while they do not influence the nodulation and N accumulation. However, the use of these herbicides in mixture can negatively influence the early nodulation and N fixation by native rhizobia.