Background: Snake venom is a complex mixture of biologically active substances. Some peptides and proteins from snake have already demonstrated their therapeutically potential. The venom of Naja haje, an Elapidae member, has been analyzed from this point of view. Understanding the fully biochemical role of its enzymes has determined the scientists to find new separation and identification methods. Objective: Our goal was to develop an optimal HPLC analytical method for separation and identification of Naja haje snake venom components, known for its neurotoxic activity. In addition, we wanted to find out if crude snake venom could inhibit the development of both Gram-positive and Gram-negative bacterial cultures. Materials and Method: Analysis of venom was performed on a HPLC system using a C16 column with UV detection at 210 nm. The analysis was done using two mobile phases, containing different concentrations of acetonitrile and trifluoroacetic acid aqueous solution at different pH values. An elution gradient was set at a flow of 0.60 mL/min. Bactericidal activity was quantified by measuring inhibition diameter around an aseptically disk filled with crude venom using Staphylococcus aureus and Escherichia coli. Results: An optimal HPLC analytical method has been developed by changing different parameters such as the pH value of mobile phase A or the elution gradient. The best resolution were obtained at a pH value of 7.4, in gradient varying from 5% to 45% in mobile phase B. Microbiological studies of the venom showed that Gram-positive bacteria growth was inhibited by crude venom, while on Gram-negative bacteria growth no effect was observed. Inhibition zone is dose-dependent and fresh crude venom is with 30% more potent than venom freeze and kept at -55°C. Conclusions: A comprehensive catalog of venom composition may serve as a starting point for studying structurefunction correlations of individual toxins for the development of new research tools and drugs of potential clinical use.