Objective: To isolate and evaluate the cytotoxic activity of different actinomycetes species isolated from the Red Sea coast in Sharm el-Sheikh, Egypt. Methods: Forty actinomycetes strains were isolated from different sediments and seawater samples collected from the Red Sea coast in Egypt. Actinomycetes were recognized by morphological and microscopic examinations. Cell viability and cytotoxicity induced by the crude extracts on breast cancer cell lines MDA-MB-231 were assessed using methylene blue assay. The strains with promising cytotoxic activity were identified by sequencing and amplifying the 16S rRNA genes. The antibacterial activities of the crude extracts were performed using Kirby-Bauer disc diffusion method. Results: The results indicated that five ethyl acetate extracts exhibited cytotoxicity towards breast cancer cell lines MDA-MB-231. The highest cytotoxic activity was found for the ethyl acetate extracts of EGY2 and EGY39. The isolate EGY3 was identified as a new Streptomyces species, while the actinomycete EGY22 was found to be a member of the genus Nocardiopsis sp. The crude extract of the isolate EGY8 showed slightly high antimicrobial activity against different test microorganisms. Conclusions: The results of the present study reveal that marine sediments of the Red Sea are a potent source of novel species of actinomycetes. The isolates may be useful in discovery of novel bioactive compounds and an important step in the development of microbial natural product research.