Pulsed Electric Field (PEF) is an emerging nonthermal food processing technology being effective in microbial inactivation without impairing the food quality. This paper demonstrates characterization of microbiota present in liquid food so that effective range of PEF can be set accordingly thus depicting the type of microorganism present, its morphology and its cell wall chemistry. The investigated microorganisms included E. coli, Staphylococcus aureus and Listeria monocytogenes, isolated from spoiled orange juice samples. These were characterized through a series of biochemical tests following microscopic (SEM) & spectroscopic (FTIR) characterization. The results were confirmed with respect to Standard MTCC (Microbial Type Culture Collection) strains (MTCC 614, MTCC 96, and MTCC 657). In comparison to Gram negative bacteria, the Gram positive bacteria are more resistant towards PEF. Larger microbial cells require less intense field strength to undergo an equivalent inactivation as compared to smaller cells. Cells in the exponential growth phase are more sensitive than the cells in lag or stationary phase, so it is necessary to identify the predominant bacteria in particular liquid foods.