Aims: The development of new antimicrobial agents towards multidrug-resistant bacteria is one of the most significant challenges facing the healthcare system today. The continuous increase of antimicrobial resistance rates worldwide is a significant threat to public health. Therefore, this study aimed to investigate the antibacterial effect of filamentous macroalgae Cladophora sp. The sample was collected from an Algerian fountainhead of fresh water. A crude hydromethanolic extract (methanol-water) was tested against two standard Gram-negative bacteria: Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853, and two standard Gram-positive bacteria: Staphylococcus aureus ATCC25923 and Enterococcus faecalis ATCC 2921. Methodology and results: The antibacterial effect of the hydromethanolic extract of Cladophora sp. was investigated using the well diffusion method to determine the inhibitory diameters and the dilution methods to determine the minimum inhibitory concentration (MIC) and the bactericidal inhibitory concentration (MBC). The results indicated that the hydromethanolic extract of Cladophora sp. is more effective towards Gram-positive bacteria, with a significant effect on S. aureus ATCC 25923, where an inhibitory diameter of 35 mm was recorded. For the Gram-negative, E. coli ATCC25922 was more susceptible with inhibitory diameters of 46 mm, followed by P. aeruginosa ATCC 27853 with 28 mm. The MIC value of hydromethanolic extract of Cladophora sp. was 50 µg/mL for Gram-positive bacteria (S. aureus ATCC25923 and E. faecalis ATCC29212). However, it was 100 µg/mL for Gram-negative bacteria (E. coli ATCC25922 and P. aeruginosa ATCC 27853). The best bactericidal effect was observed with Gram-positive with an MBC of 100 µg/mL. The MBC for Gram-negative bacteria was 150 µg/mL. Conclusion, significance and impact of study The Cladophora sp. macroalgae represent a potential source of bioactive compounds, which could be used in the management and treatment of various microbial infections.