Urease is a key virulence factor for the urinary tract pathogen Staphylococcus saprophyticus. Previous studies indicated that dimethylsulfoxide, which resembles urea, could inhibit urease activity in a noncompetitive fashion but could not completely prevent the increase in pH associated with urease activity. The aims of this study are 1) to investigate the effects of boric acid, sodium fluoride, phenylmethylsulfonyl fluoride and dimethylglyoxime on the urease from S. saprophyticus and 2) to determine if these compounds in combination with dimethylsulfoxide could better inhibit bacterial growth and pH changes in artificial urine medium and human urine. Urease activity in extracts and whole cells was measured by the formation of ammonium ions. The effects of urease inhibition were determined by following bacterial growth, pH, and formation of insoluble crystals in cultures grown in artificial urine medium, urea broth, and human urine. Boric acid competitively inhibited urease activity in cell extracts up to 80% while sodium fluoride reduced activity up to 90% in an uncompetitive fashion. Phenylmethylsulfonyl fluoride inhibited activity about 80% but dimethylglyoxime only reduced activity by 50%. All of the compounds inhibited urease activity in whole cells grown in artificial urine medium. Boric acid and sodium fluoride in combination with dimethylsulfoxide blocked the color change that occurs in urea broth. When boric acid, sodium fluoride, and dimethylsulfoxide were added to S. saprophyticus cultures in artificial urine medium, there was a longer lag phase, a decrease in the rate at which the pH rose, and an inhibition of struvite crystal formation. These compounds also reduced bacterial growth and the pH increase in normal human urine. A combination of active-site inhibitors thus can reduce the physiological effects of urease activity and may be effective in treating patients with urinary tract infections that are resistant to antibiotics or herbal remedies.