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Pistacia integerrima Stew ex Brandis is a valued medicinal plant used for curing various diseases such as diarrhea, fever, liver disorder, pain, asthma, and inflammation. The aim of this study was the isolation of bioactive leishmanicidal agents from the methanolic extract. The methanolic extract led to the isolation of flavonoids 3,5,7,4/-tetrahydroxy-flavanone (1). The extract and isolated compound 1 were tested for antileishmanial effect. The extract showed a percent effect of 63.09 with an IC50 value (49.32 µM). The isolated compound 1 was more leishmanicidal than the extract with a percent growth inhibition of 68.09. We have performed docking studies on two antileishmanial targets; homology modeled dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) from Leishmania major (L. major). Interaction with important residues of the studied enzymes revealed the possible mechanism of in-vitro activity against promastigotes of L. major.