Background and objectives: In the resent years, using different kind of herbal extracts had been considered for bacterial infection. Sedr or Konar which scientific name is Ziziphus spina-christi L. is native to tropical and subtropical regions. This study aimed to evaluate the presence of functional groups and antimicrobial activity of Ziziphus spina-christi aqueous extract on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria innocua “ in vitro” . Materials and methods: In this laboratory study, extraction of ziziphus spina-christi L. leaf powder with maceration method was done for 48 hours. Leaf extracts antibacterial of these leaves were evaluated by four methods: Disc diffusion agar, well diffusion agar, minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC). The functional groups of Ziziphus spina-christi aqueous extract was evaluated by Fourier transform infrared spectroscopy (FTIR). Results: In disc diffusion agar and well diffusion agar methods, all the concentration of ziziphus spinachristi L. restrained bacterial strains. The MIC was for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria innocua about 64, 32, 8 and 16 respectively. The results showed that MIC of extracts was further of MBC. The main peaks of Ziziphus spina-christi aqueous extract factor groups were observed in wave numbers of 3390, 2974, 2933, 1715, 1606, 1419, 1307, 1274, 1059, 885, 820, 779 and 629 cm. Conclusion: Based on results, the Ziziphus spina-christi aqueous extract had desirable antibacterial effect. The results showed that Gram-negative bacteria showed further resistance against extract. Using this herbal compound could be effective to control pathogenic bacteria in food and pharmaceutical industries.