In this study, total phenolic and flavonoid content as well as in vitro antioxidant and antimicrobial activity of the methanolic extract of Ziziphus lotus were evaluated using different methods. The total phenol and flavonoid content in the extract were determined by Folin Ciocalteu and AlCl3 assays, while antioxidant and antibacterial activities were studied using DPPH free radical-scavenging, ABTS, disc diffusion and micro-dilution methods. The results showed that the phenols amount (143.12 mg/mL) in the methanolic extract of Ziziphus lotus were thirty-five fold higher than the flavonoids (4.28 mg/mL). Furthermore, Ziziphus lotus showed higher antioxidant potency as determined by DPPH and ABTS methods, (DPPH IC50 values of 131.01 µg/mL compared to 50.67 for BHT, and ABTS IC50 values of 52.42 µg/mL compared to 63.44 µg/mL for Trolox). Accordingly, antibacterial tests showed that the methanolic extract exhibited a powerful antibacterial effect against the bacterial strains with MIC values of 400 and 320 µg/ml against E.coli and Bacillus pumilus, respectively. These results indicated that Ziziphus lotus fruit could be taken as a potential source of phenolic compounds well-known for their impact in human health as well as nutrition.