Background: Among marine organisms, seaweeds are a highly diverse group of organisms from which many new substances have been isolated and many of these compounds have been demonstrated to possess a large spectrum of bioactivities. Objective: In this study, we aim to evaluate the antibacterial, cytotoxic and antioxidant activity of green algae, Cladophora prolifera (Roth) Kutzing, collected from the northern Mediterranean coast of Morocco. Material and Method: The antibacterial activity was determined by disk diffusion method. After fractionation by column chromatography, the fractions from C. prolifera were tested against Staphylococcus aureus ATCC 25923 using the broth microdilution assay. The antitumor effect on human colon cancer cells was investigated via sulforhodamine-B (SRB) assay. Then the Crude extract has been tested in radical-scavenging assays to assess their antioxidant activity. Results: In vitro screening of methanolic extract of C. prolifera showed specific activity to inhibit the growth of five virulent strains of pathogenic bacteria, Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212) and Klebsiella pneumoniae (ATCC 700603). The obtained results indicated that the extracts of C. prolifera were cytotoxic against HT29 human colon cancer cells. In addition, based on the capacity of the algae to scavenge the ABTS radical cation, we revealed that C. prolifera extract presented a satisfactory antioxidant activity.