Sorgoleone is a secondary sorghum metabolite released from roots. It has allelopathic properties and is considered to inhibit ammonia-oxidizing archaea (AOA) and bacteria (AOB) responsible for the rate-limiting step (ammonia oxidation) in nitrification. Low activity of these microorganisms in soil may contribute to slow down nitrification and reduce nitrogen loss via denitrification and NO 3 − leaching. The potential nitrification rate (PNR) and the composition of microbial communities were monitored in rhizosphere soil to investigate the growth effect sorghum on biological nitrification inhibition (BNI). A greenhouse pipe experiment was conducted using sorghum lines IS20205 (highsorgoleone release ability), IS32234 (medium-sorgoleone release ability), 296B (low-sorgoleone release ability), and a control (no plants) combined with fertilization application of 0 or 120 kg N ha −1. We applied nitrogen as ammonium sulfate at 16 days (20 N), 37 days (40 N), and 54 days (60 N). We collected soil solutions at 7.5 cm depths every 3 days and measured the pH and nitrate levels. At 1 and 2.3 months, we sampled the bulk and rhizosphere soils and roots in the 0-10 cm, 10-30 cm, and 30-80 cm depths to determine NO 2 , mineral N, total N, total C, sorgoleone, the composition of AOA, AOB, and total bacteria and archaea. Sorgoleone was continuously released throughout the 2.3 months' growth and was significantly higher in IS20205, followed by IS32234 then 296B, which showed shallow levels. The IS2020 5rhizosphere showed lower NO 2 and nitrate levels and significant inhibition of AOA populations. However, we did not find significant differences in the abundance of AOB between plant treatments. Multivariate analysis and Spearman's correlations revealed that sorgoleone as well as environmental factors such as soil pH, soil moisture, NO 3 −-N, and NH 4 +-N shape the composition of microbial communities. This study demonstrated that the release of higher amounts of sorgoleone has great potential to inhibit the abundance of AOA and soil nitrification. The breeding of sorghum lines with the ability to release higher amounts of sorgoleone could be a strategic way to improve the biological nitrification inhibition during cultivation.