Diallyl disulfide (DADS) is an organosulfur compound that is expected to exhibit inhibitory property against urease similar to allicin, affirmed through preliminary study. The research aims to optimize DADS�s concentration and duration of inhibition and observe the effect of soil moisture, temperature and pH on the inhibitory action of DADS. The calorimetric method was applied to optimize DADS�s concentration significant for inhibition. High-performance liquid chromatography was used to quantify DADS present under different parameters relevant to selected soil conditions. The results obtained suggested that 5 of DADS/urea-N (w/w) treatment exhibited the highest urea hydrolysis reduction by 27.91 compared to the control sample at the end of 30 days. ANOVA results observed urea hydrolysis is significantly slower by applying 5 DADS/urea-N (w/w) treatment compared to the other DADS treatments. DADS also retained its original form longer in soil when the soil conditions were altered to 15 moisture content, 20 °C and pH 4. The findings exhibit the potential of DADS as a natural based inhibitor that is effective at low concentrations, compatible with urea and chemically stable. © 2020, Czech Academy of Agricultural Sciences. All rights reserved.