Introduction: It was reported that snake venom contains a wide mixture of proteins and peptides with various toxicological and pharmacological effects. In recent years, the isolation of effective molecules from snake venom as a drug to treatment of some incurable diseases is considered by many biological research centers. This investigation aimed to determine electrophoretic and chromatographic patterns of Pseudocerastes persicus snake venom and in-vitro study of bactericidal and anti-cancer properties of its fractions in a human hepatocellular liver carcinoma cell line (HepG2). Materials and methods: The fractions of venom were isolated and collected using preparative RP-HPLC. The Bactericidal activity of fractions in 20 μg/ml of protein concentration was investigated toward Gram-negative and Gram-positive bacteria. Antibacterial susceptibility tests were done using MTT and MIC assays. Furthermore, cell viability was measured by MTT reduction assay and confirmed with Neutral red uptake assay following exposure of HepG2 cancer cells to 20 and 40 μg/ml of protein concentrations of fractions having more antibacterial properties. Apoptotic effects were investigated using the comet assay. Results: SDS-PAGE pattern of the crude venom revealed 10 major bands with molecular weight ranging from 13.2 to 99.25 kDa and the single-step separation of different fractions from the venom by HPLC resulted in isolation and collection of 11 fractions. The results of this study showed that two fractions of snake venom have significant antibacterial activity against Gram-positive as compared to Gram-negative bacteria. On the other hand, our findings indicated that one of them is not toxic to the HepG2 cells. Discussion: We for the first time reported bactericidal and anticancer effects of isolated fractions of Persian horned viper venom. These properties make components of these fractions a favorable source for isolation of molecule(s) with antibacterial and antitumor activities.