Production of antitumor agent(s) from alternative natural marine microbial resources may decrease or avoid the bad side effects of the used anti-tumour chemotherapeutic agents and may increase the specificity of the antitumor agents to be safer for human application. So, this study aimed to search for new alternative antitumor agent producers using two recommended bioassays (Red Potato Disc Bioassay and Sulforhodamine B Bioassay). The obtained results indicated the isolation of three new marine actinomycetes strains from the western harbor of Alexandria, they identified using the 16S rRNA gene sequence as Streptomyces sp. strain AMS11, Nocardiopsis halotolerans strain AMS10b and Nocardiopsis halotolerans strain AMS10a. The bio-toxicity test was carried out using the biomarker Artemia salina, they showed a moderate toxicity level (LC 50 <1000 ppm). The antitumor activities of their supernatants against the colon cancer cell line (HCT116) and the liver cancer cell line (HEPG2) showed promising results in comparison to the recommended chemotherapeutic drug 5-flourouracil used for the treatment of both liver and colon cancer cells. The optimization for antitumor activity was performed using the Plackett -Burman experimental design. [6,7]. The bio-toxicity of the potent supernatants was performed using the biomarker Artemia salina [8]. Finally, the optimization to reach the ideal conditions for maximum bioactivity was carried out using a Plackett-Burman experimental design [9]. Production of Antitumor Agents from Novel Marine Actinomycetes Strains Materials and Methods Isolation of different actinomycetesIsolation of different marine actinomycetes was carried out from 10 stations of the western harbor of Alexandria, Egypt. Four recommended actinomycetes culture media; Oatmeal (shofan) Nitrate Medium, Starch Casein Nitrate Medium, Starch Nitrate Medium and Potato Dextrose Medium were tested in order to select the most suitable culture medium for antitumor production [10][11][12]. The supernatant of each actinomycetes isolate was obtained using a micro-centrifuge for 15 min at 12000 rpm then undergo antitumor activity test. Screening for the antitumor activity Red Potato Disc bioassayThroughout this assay the potato discs has been infected by a tumor inducer bacterium "Agrobacterium tumefaciens". The examined potato discs were inoculated with 50 µl/disc of; B: sterile seawater, C 1 : A. tumefaciens suspension with OD=1 (the bacterial infection effect), C 2 : A. tumefaciens+1% Ampicillin (the T iplasmid effect only) and T: C 2 +the supernatant (1:2) of the tested actinomycetes The positive antitumor activity of each supernatant was detected by the results of five examined potato discs as replicates and confirmed through two successive experiments. All discs were incubated at 28°C for two weeks. The antitumor activity % was calculated according to Coker et al. [13] and El-Masry et al. [14]. The anti-tumor activity percentage was calculated after flooding the discs with the Gram iodine solution for one min. The ...