The influence of solvent extractions (ethanolic and hydroethanolic extract) and extraction by controlled differential sieving processing (CDSp) on chemical content, in vitro and in vivo antioxidant capacity of Diospyros mespiliformis fruits powders was investigated. D. mespiliformis fruits powder were finely ground and fractionated by CDSp into four particle size fractions (< 50 μm; 50 -180 μm; 180 -315 μm; and > 315 μm). The chemical components determined were carotenoids, vitamin C, vitamin A and saponin. The in vitro antioxidant activity was carried out using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. To evaluate the in vivo antioxidant activity against high fat diet (HFD) induced hyperlipidemia in rats; the different particle size powder fractions, ethanolic and hydroethanolic extract of D. mespiliformis fruits were administered orally (600 mg/kg, po) for 30 days, with HFD and the effect of extract on enzymatic antioxidants like superoxide dismutase (SOD), catalase (CAT) and peroxidase were estimated in the blood, heart, liver and kidney. Toxicity parameters like alanine transaminase and aspartate aminotransferase were evaluated in these organs. The ˂ 50 µm fraction contained higher carotenoids (19.27 ± 0.21 mg/100 g DW) and vitamin A (3.21 ± 0.03 mg/100 g DW) than the other fractions, unsieved powder (6.87 ± 0.11 mg/100 g DW for carotenoids and 1.15 ± 0.02 mg/100 g DW for vitamin A) ethanolic extract (7.02 ± 0.30 mg/100 g DW for carotenoids and 1.17 ± 0.04 mg/100 g DW for vitamin A) and hydroethanolic extract (3.56 ± 0.19 mg/100 g DW for carotenoids and 0.59 ± 0.03 mg/100 g DW for vitamin A) of D. mespiliformis fruits. The vitamins were highly concentrated in ethanolic extract (12.22 ± 0.02 mg/100 g DW) followed by powder fraction of < 50 µm (11.11 ± 0.41 mg/100 g DW). The different samples of D. mespiliformis fruits powder improved the SOD, catalase, peroxidase, alanine transaminase and aspartate aminotransferase levels significantly as compared to the control group. The present study has revealed that the fraction, < 50 μm powder of D. mespiliformis fruits have significant in vivo antioxidant activity than the ethanolic extract and can be used to protect tissues from oxidative stress.