Background Due to the important economic role of pistachio (Pistacia vera L.) the cultivation of this valuable crop has been extended. Various abiotic stresses harm the growth and performance of pistachio. Seaweed extract containing various substances such as pseudo-hormones that stimulate growth, nutritional elements, and anti-stress substances can cause more resistance to abiotic stresses, and increase the quantity and the quality of the fruit. The present study was conducted to evaluate the effect of foliar application of Ascophyllum nodosum (L.) Le Jol. seaweed extract on some biochemical traits related to abiotic stress in Pistacia vera L. cv. Kaleh-Ghoochi. The first factor of foliar spraying treatment included A. nodosum seaweed extract at four levels (0, 1, 2, and 3 g/L), and the second factor was the time of spraying solution which was done at three times (1- at the beginning of pistachio kernel growth period at the end of June, 2- at the stage of full kernel development at the end of August, and 3- Spraying in both late June and August). Results The results showed that all investigated traits were significant under the treatment of seaweed extract compared with the control. The seaweed extract concentrations had a significant effect on all traits except soluble carbohydrates, but the time of consumption of seaweed extract on soluble carbohydrates, protein, peroxidase, ascorbate peroxidase, and superoxide dismutase enzymes was significant, while had no significant effect on the rest of the traits. According to the interaction effect of time and concentration of consumption of seaweed extract, the highest values of the biochemical characters were as follows: total phenol content: 168.30 mg CAE/g DW, flavonoid content: mg CE/g DW, catalase: 12.66 μmol APX min− 1 mg− 1 protein, superoxide dismutase: 231.4 μmol APX min− 1 mg− 1 protein, and ascorbate peroxidase: 39.53 μmol APX min− 1 mg− 1 protein.