Microalgae are recognized as promising sources for the development of pharmaceutical compounds and the discovery of natural antibiotics to combat antibiotic resistance, due to their ability to produce a wide range of bioactive metabolites. However, despite limited reports on the biological activities of microalgae, comprehensive and systematic data regarding the phytochemical composition and the antioxidant and antibacterial activities of the green alga Zygnema, particularly in its natural habitats in northern Iran, remain insufficient. The aim of this study was to evaluate the antioxidant activity using the DPPH method and the antibacterial activity of phytochemical compounds in the methanolic extract of the green alga Zygnema collected from aquatic habitats in northern Iran against two common human pathogenic bacteria, Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative), using the agar well diffusion method. Phytochemical screening and GC–MS analysis showed that the methanolic extract of Zygnema contained hexadecanoic acid methyl ester, methyl linolenate, phytol, gamma-sitosterol, and isofucosterol, as well as phenolic, flavonoid, carotenoid, chlorophyll, and lipid-derived compounds, exhibiting strong antioxidant activity (81%). Antibacterial evaluation revealed that the methanolic extract showed the highest inhibitory effect against S. aureus with a maximum inhibition zone of 32 mm, while an inhibition zone of 23 mm was observed against E. coli. In conclusion, due to the presence of effective bioactive compounds, the methanolic extract of the green alga Zygnema exhibits significant antioxidant and antibacterial activities and may be considered a promising multi-target natural agent with potential application in therapeutic strategies.