Salinity is an important challenge around the world, effecting all physiological and biochemical processes of plants. It seems that seed priming can diminish the negative impacts of salinity. To study the effects of hydro-priming and inoculation with Piriformospora indica (Pi) and Azotobacter chroococcum (Az) on physio-biochemical traits, flavonolignans and fatty acids composition of milk thistle under saline conditions, a greenhouse experiment was carried out. Our results indicated that under salinity, seed priming, especially Pi, improved physio-biochemical properties in milk thistle. Under 120 mM NaCl, inoculation with Pi increased membrane stability index (MSI) and relative water content (RWC) (by 21.86 and 33.43%, respectively). However, peroxidase (POX) (5.57- and 5.68-fold in roots and leaves, respectively), superoxide dismutase (SOD) (4.74- and 4.44-fold in roots and leaves, respectively), catalase (CAT) (6.90- and 8.50-fold in roots and leaves, respectively) and ascorbate peroxidase (APX) (5.61- and 5.68-fold in roots and leaves, respectively) activities increased with increasing salinity. Contrary to salinity, hydro-priming with Az and Pi positively altered all these traits. The highest content of the osmolytes, adenosine triphosphate (ATP) content and rubisco activity were recorded in Pi treatments under 120 mM NaCl. Stearic acid (20.24%), oleic acid (21.06%) and palmitic acid (10.48%) increased, but oil content (3.81%), linolenic and linoleic acid content (22.21 and 15.07%, respectively) decreased under saline conditions. Inoculations of Pi positively altered all these traits. The present study indicated that seed priming with Pi under 120 mM NaCl resulted in maximum silychristin, taxidolin, silydianin, isosilybin, silybin and silymarin of milk thistle seeds.