This paper conducts green synthesis of silver nanoparticles using Pistacia palaestina fruit extract and studies the effects of the fabrication conditions on the size of fabricated nanoparticles during the synthesis process and the evaluation of its wound healing activity in vivo. Methods: P. palaestina extract has been used to reduce silver nitrate solution to AgNPs. The synthesized AgNPs were characterized using UV-Vis spectroscopy, Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), and Scanning Electron Microscopy (SEM). The fabricated AgNPs were prepared as an ointment (1% w/w) and evaluated for wound healing activity using an incision wound model on rats. The time of healing was determined and compared with a positive control (gentamicin ointment 0.1%) and a negative control. Results: The fabricated AgNPs have a dark brown color, and their absorption peak was at 430 nm. The obtained particles have an average diameter of 36 nm by SEM, 55.64 nm by DLS, and 100 nm by AFM. AFM and SEM images confirmed the spherical shape of prepared AgNPs. The optimal formula for preparing AgNPs was by using fruit extract (pH 7) at a ratio of (1:9) at 35°C for 24 h. The prepared AgNPs ointment (1%) accelerated wound healing compared to a negative control group, as the time of healing was 9 ± 1.41, 12 ± 1.73, and 17 ± 2.00 days, respectively. Conclusion: It is concluded that P. palaestina extract can be utilized for fabricating AgNPs without any additions, and they have wound healing effect in experimental rats.