Cladophora glomerata has numerous biologically active properties and is considered one of the most essential medicinal algae. The purpose of this research was to investigate the anticancer efficacy of Cladophora glomerata algae extract against human hepatocellular carcinoma (HepG2), human cervical carcinoma (HeLa), and normal mouse embryonic fibroblast (MEF) cell lines. The needed algae was found in the Hassan Al-Hamoud River , Baquba, Iraq. The chemical detection of some chemical components of the ethanolic extract of C. glomerata revealed that the extract contains a group of active compounds. The study showed significant variation (p<0.05) among inhibition percentages of cancer cell line HepG2, Hella, and MEF cell lines that were treated with different concentrations (15.1, 31.2, 62.5, 125, 250, 500, and 1000) μg/ml of C. glomerata extract. The outcomes showed that increased inhibition percentages of the above cell line were associated with increased concentrations. The inhibition percentage of HepG2, Hella, and MEF cell lines were 1.6 ± 30.88, 1.7 ± 14.10, and 1.2 ± 2.31 at concentration 15.1 (mg/ml), and was 3.8 ± 84.90, 3.6 ± 88.29, and 3.9 ± 23.2, respectively at concentration 1000 (mg/ml). The study concluded that the C. glomerata extract with different concentrations significantly inhibited cancer cell lines (HepG2 and Hela) and ME because they have antiproliferative and antioxidant activity against cancers. The extract's inhibitory impact begins at low doses and increases with increasing concentration. The study would be beneficial to use macroalgae as new and sustainable sources of bioactive compounds against cancer.