Urease inhibitor is considered as one possible method for increasing the utilization efficiency of dietary urea in ruminant animals. Previous studies suggest that Cys319 located on the Thr308-Arg336 flexible loop covering the active site of Klebsiella aerogenes urease could be used as a targeting point to inhibit urease activity. In this study, we employed a structure-based molecular docking of the Chemdiv database against the Thr308-Arg336 flexible loop of K. aerogenes urease (PDB:4EP8). The Chemdiv database was prepared by using the Compound Filtering module of sybyl-X2.1 software. Two rounds of virtual screening were implemented. Eventually, 99 compounds were achieved to undergo in vitro screening against rumen bacterial urease, which performed by the Berthelot alkaline phenol–hypochlorite method. The IC50 values were determined by nonlinear curve fitting of gradient concentration of the compound with residual activity of urease. Sixteen compounds were found to be relatively potent urease inhibitors with the inhibition rate over 40% at the concentration of 0.5mM. The compound 3-[1-[(aminocarbonyl)amino]-5-(4-methoxyphenyl)-1H-pyrrol-2-yl] propanoic acid (compound 22) was found to be superior agent in the series with an IC50 = 68.42μM. The binding mode of compound 22 was revealed by molecular docking study. Three hydrogen bonds are formed between compound 22 and important active site residues; Asp221, Arg336 and Asp220. Two hydrogen bonds are also formed between the carboxyl group of compound 22 and important active site residues; Ala363 and Asp360. Relatively strong hydrophobic interaction is formed between compound 22 and hydrophobic residues; His320, Cys319, Met364 and Ala167. This study reaffirmed the viability of using the Thr308-Arg336 flexible loop covering the active site of urease as a target to screen chemical databases. These compounds can be used as starting points for lead optimization.