To determine the novelty and potency of the compound with antibacterial activity minimum inhibitory concentration (MIC) was studied against four different Gram negative bacteria using the broth dilution assay. For this purpose test compound Di-2-ethylaniline phosphate was prepared in the range from 50-10,000 μg/ml in dimethyl sulfoxide. Highest percentage growth inhibition in terms of minimum inhibitory concentration was found when 2 ml of 600 μg/ml concentration was used against Gram negative bacteria. Decreasing optical density was reported with increasing concentration of test compound. So it has been concluded that the minute concentration of Di-2- ethylaniline phosphate have the antibacterial activity with high potency.