A strain of Pseudovibrio sp. strain 4MS 2020 with high exopolysaccharide production was isolated from sawa lake in Iraq and identified based on morphological, physiological, biochemical characteristics and phylogenetic examination of 16S rDNA sequences. Pseudovibrio sp. strain 4MS 2020 was optimized for production of maximum yeild of EPS which gave 10.8 g/l after 3 days of incubation with pH 7, 40°C, RPM 150, 750 µl inoculum sizes, yeast extract as nitrogen source. While, 20 g/l sucrose as a carbon source. EPSS2 was a major fraction of EPS contained sulfate (53.9%) and has no uronic acid. The monosaccharide composition of these fraction composed of glucose, rhaminose and arabinose with molar ratio 6.0 : 3.0 : 1.0, respectively. Then EPSS2 was Prepared as Nanocrystalline Exopolysaccharide (NCEPSS2).Therefore, EPSS2 and NCEPSS2 were subjected to antioxident and anticancer activities, scince it showed that EPSS2 give maximum activity against DPPH radical found 88.8 % at 1500 µg/ml after 120 min, while against H2O2 radical found that the activity was 64.8 at 1500 µg/ml after 60 min. while, NCEPSS2 give antioxidant activity was 99.3 % at 1500 µg/ml after 120 min. and the maximum antioxidant activity was 81.6 % at 1500 µg/ml after 60 min. for H2O2 radical. By using MTT assay, EPSS2 and NCEPSS2 was effect on the proliferation of HCT-116, A-549, HepG-2 and MCF-7 cells. the calculated IC50 for EPSS2 at cell lines HCT-116 was 230 µg/ml and HepG-2 showed a IC50 values 203 µg/ml. While, the calculated IC50 for cell line MCF-7 and A-549 showed no cytotoxic level for these cell lines. While, NCEPSS2 calculated IC50 for cell line HCT-116 was 119 µg/ml. While, the cell line HepG-2 showed a IC50 values 107 µg/ml. IC50 values of cell line A-549 showed 97.7 µg/ml and the calculated IC50 for MCF-7 was 200 µg/ml