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The urease inhibitor N -(n -butyl) thiophosphoric triamide (NBPT) has been reported to effectively reduce nitrogen (N) losses by inhibiting urea hydrolysis. However, the effect of NBPT on soil ureolytic and ammonia-oxidizing microbes is not well understood, with inconsistent effects on crop yield and nitrogen use efficiency (NUE). Further, repeated use or overuse of chemical fertilizers can have environmental implications like soil acidification. A field experiment was conducted at Leigh Creek, Victoria at a site with a history of repeated application of urea alone (40U) or with NBPT (as Green Urea NV® (40GU)) at 40 kg N ha−1, and urea applied at 80 kg N ha−1 (80U), to perennial ryegrass (Lolium perenne L.). We aimed to investigate the temporal effects of NBPT applied with urea on ureolytic and ammonia-oxidizing microbes, pasture dry matter (DM) yield, and NUE within a season following treatment applications. The abundance of ureolytic microbes was higher in the control (CK) compared to all N treatments on all sampling days. The ureC gene copy numbers in 40 GU were significantly lower than in 40U on sampling days seven and 45. There was no significant effect of NBPT on the abundance of ammonia oxidizers, but increasing urea application rate to 80 kg N ha−1 (80U) significantly increased the abundance of ammonia-oxidizing bacteria (AOB) on days 7 and 45, and complete ammonia oxidizers (comammox Nitrospira) clade B on day 7 compared to 40U. There was no significant effect of NBPT on pasture DM yield, N-uptake, or NUE. Increasing N application rate significantly increased pasture DM yield and N- uptake but this did not influence the pasture NUE. [ABSTRACT FROM AUTHOR]