Abstract : Objective To assess anti-allergic conjunctivitis effect of hydroalcoholic extract of Moringa oleifera (HMO) and its isolated constituents in rats. Methods The acute oral toxicity study of extract was carried out up to the dose level of 2000 mg/kg for HMO and 300 mg/kg for constituents (Myricetin and Kaempferol) by following OECD guideline no. 423. On the first day, allergic conjunctivitis was induced by injecting (i.p.) 0.6 ml of physiological saline that included egg albumin (1 mg), alum (2 mg), and 1010 dead Beta pertussis cells. After five days, they were given a boost through subcutaneous administration with one ml of physiological saline containing 0.5 mg of egg albumin at ten different locations on the back. Then from days 14th to 42nd the rats were treated with hydroalcoholic extracts of Moringa oleifera (HMO) according to their respective group at doses of 100 mg/kg, 200 mg/kg and 400 mg/kg p.o. and isolated constituents, Myricetin 100 mg/kg, Kaempferol 100 mg/kg while the standard group was treated with Cetirizine hydrochloride at doses of 10 mg/kg p.o. in comparison with control group who receive 1% w/v CMC solution. Following administration of the dose, a local sensitization procedure was carried out by injecting egg albumin diluted in physiological saline at a concentration of 10 mg/ml using a micropipette into the bilateral eyes. Then allergic signs and eye scratching behaviour was observed. Diethyl ether was used to induce anaesthesia in the rats, and their conjunctivas were removed 24 hours after being challenged on days 14, 21, 28, 35, and 42. The animals were also sacrifice during this process. The tissues were fixed in 10% neutral buffered formalin for two days before being examined. After a period of 2 days, the samples were paraffin-fixed, and conjunctiva tissues that were 4 micrometres thick were stained in order to determine number of eosinophils in the conjunctiva. Results During the acute and sub-acute oral toxicity study of extract and its constituents, no mortality was observed in any animals up to the dose level of 2000 mg/kg for HMO and 300 mg/kg for constituents (Myricetin and Kaempferol), indicating their practically nontoxic in nature. Oral administration of extract significantly decreased allergic signs and eye scratching behaviour (p<0.001) and markedly decreased eosinophil into the conjunctival tissues (p<0.01) in dose dependent manner. A similar effect is observed with isolated constituents as of 400 mg/kg HMO. Conclusion The present study showed that the hydroalcoholic extract of Moringa oleifera (HMO) and its isolated constituents (Myricetin and Kaempferol) produced a significant anti-conjunctivitis activity against allergic conjunctivitis as well as it is entirely safe.