In the present work, the effect of methanolic extract of bark of Acacia nilotica on nucleation and aggregation of calcium oxalate crystals and on ethylene glycol induced lithiasis was evaluated. The yield of the methanolic extract was found to be 6.21 % (7.079 g). The findings of phytochemical analysis suggest the presence of of glycosides, phenolics and tannins, flavonoids and carbohydrates in the bark of the plant. The phenolic content in the methanolic extract of Acacia nilotica was found to be 26.9 ± 1.33 % w/w. The administration of methanolic extract of Acacia nilotica bark caused an inhibition of the slope of rate of nucleation as well as the rate of aggregation of calcium oxalate crystallization. The methanolic extract was significant in preventing the formation of crystal and was further tested in animal using ethylene glycol induced lithiasis. The methanolic extract was able to exert diuretic action in rats and also decreased the activity lactate dehydrogenase and alkaline phosphatase, the biomarkers of the biomarkers of lithiasis.