Astaxanthin is a valuable carotenoid which has been approved as a food coloring by the US Food and Drug Administration. This work aimed to attain Rhodosporidium toruloides mutants for enhanced astaxanthin accumulation using ethyl methanesulfonate mutagenesis (EMS). EMS treatment was shown efficient at different investigated concentration included 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 6.0% in affecting the yeast for enhanced astaxanthin production. Among the screened mutants, E4 (1719.9 µg/L), an EMS-induced mutant at 3.5% concentration, exhibited the highest astaxanthin production which was significantly higher than that of the wild strain (increased 4.7 times). A mutant yeast strain E4 showed a short lag phase of about 2 hours, then 32 hours of log phase, 35 hours of stationary phase and then entered to death phase after 70 hours of inoculation. The best time to collect astaxanthin was after 96 hours of inoculation in Hansen broth, the accumulated astaxanthin content was 1719 µg/L. Crude extract of astaxanthin from mutant strain E4 had the scavenging capacity of DPPH free radicals (IC50 = 9.106 µg/mL) 10.6 times higher than that of vitamin E and their’s application direction in the pharmaceutical industry, nutritional supplements, as well as bioproduct used in the aquaculture industry.