Distillery spentwash (DSW) R.O. Reject is a dark brown coloured liquid which contains major and micro-nutrients which are helpful for sustaining the soil fertility and the yield of crops. The dark brown colour of raw spentwash is due to the presence of melanoidin of cane molasses which is not decomposed effectively by yeast and methane bacteria in its activated sludge process. A field experiment was conducted to see the effect of this distillery spentwash R O reject on soil organic carbon content and enzymatic activity in soil during kharif 2013. The experiment consisted of seven treatments laid out in Randomized Complete Block Design with three replications. The experimental results indicated that the application of 150% N through distillery spentwash (DSW) R O reject increased the soil organic carbon content (0.83%) and activity of different enzymes like phosphatase (21.3 µg P-NPP g-1 of dry soil hr-1), dehydrogenase (9.1 µg TPF g-1 of dry soil hr-1) and urease (43.6 µg NH4-N g-1 of dry soil hr-1) compared to all other treatments. Significantly lower values of soil organic carbon content and activity of different enzymes were recorded in treatment receiving RDF only. It is concluded that application of distillery spentwash R O reject results in increased organic carbon content and enzyme activities.