Finding alternative powerful antibacterial drugs of natural origins is, today, a crucial prerequisite due to the resistance of some bacterial strains to commercial and widely-used medications. Algae are characterized by their bioactive constituents and have a wide spectrum of biotechnological aspects, particularly antibacterial implications. During this study, four concentrations (5, 10, 20, and 40 mg mL–1) of the Egyptian untapped green microalga Rhizoclonium hieroglyphicum (Chlorophyta) were prepared using the polar solvents ethanol, methanol, and acetone. The antibacterial activity of the above-mentioned extracts was assessed, using the agar disc diffusion technique against three pathogenic bacteria, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and Pseudomonas aeruginosa ATCC 9027, which was compared to standard antibiotics. The minimal inhibitory concentrations (MICs) were also assessed and determined using a broth dilution assay. Our findings revealed that the R. hieroglyphicum ethanolic extract exhibited the most potent antibacterial effect and its MICs values were 0.533, 2.25, and 5.34 mg mL–1 against P. aeruginosa, E. coli, and S. aureus, respectively. A gas chromatography–mass spectrometry (GC–MS) approach to the crude R. hieroglyphicum ethanolic extract uncovered 30 different bioactive constituents, mainly including long-chain polyunsaturated and saturated fatty acids such as myristic (C14:0), palmitic (C16:0), stearic (C18:0), α-linolenic (C18:3; ω–3), and oleic (C18:1, ω–9) acids, which synergistically make this potent antibacterial action. The mechanism of action of these fatty acids was also discussed. Conclusively, R. hieroglyphicum could be a good candidate for the production and development of promising antibacterial agents.