The haemato-biochemical indices and oxidative stress markers in horses naturally infected with Trypanosoma evansi were evaluated by analyzing the level of these parameters between T. evansi infected (microscopically positive patent group and PCR positive latent group) and infection free horses. To compare the hemato-biochemical indices and oxidative stress indicators, horses were divided into three categories based on diagnostic test employed and positive results obtained. These included Romanowsky stained slide positive group (Group I; n = 6), PCR positive group (group II; n = 28) and negative control group (group III, n = 30), revealing parasitologically positive patent, molecular positive latent and disease free status of horses. A significant reductions in total erythrocytes count (TEC, P = 0.01), haemoglobin (Hb, P = 0.01) and packed cell volume (PCV, P = 0.04) was noticed both in group I and group II while significant neutrophilia and lymphocytopenia was observed in group I when compared to negative control group. Substantial increase in creatinine (CRTN, P = 0.032) and gamma glutamyl transferase (GGT, P = 0.012) in group I while significant decrease in glucose (GLU, P = 0.04) and iron (Fe, P = 0.01) were noticed in both group I and group II in comparison to group III. A significant difference in lipid peroxides (LPO, P = 0.01) with highest level in patent group I (15.33 ± 0.53) followed by PCR positive latent group (14.09 ± 1.66) indicates higher lipid peroxidation in erythrocytes and oxidative stress in decreasing order when compared with infection free control horses (9.83 ± 0.97). Catalase (CAT, P = 0.01) was significantly lower in parasitological (0.82 ± 0.14) and molecular positive cases (1.27 ± 0.35) in comparison to control group (3.43 ± 0.96). The levels of superoxide dismutase (SOD, P = 0.01), reduced glutathione (GSH, P = 0.01) and ferric reducing antioxidant power (FRAP, P = 0.01) were significantly lower in parasito-molecular positive cases as compared to infection free control horses. An inverse correlation of RBC count with LPO and GSH and a direct correlation with catalase, SOD and FRAP was revealed. Overall, the observed substantial decreases in the oxidative parameters like catalase CAT, SOD, GSH and FRAP activities with remarkably elevated levels of LPO indicate high exposure of erythrocytes to oxidative damage in T.evansi infected horses.