Eshtaol was founded on the lands of two abandoned Arab villages, Ishwa' and Islin after the 1948 Arab-Israeli War. It was part of a plan to establish settlements in the Jerusalem Corridor in order to create a contiguous bloc between the coastal plain and Jerusalem. The first residents were Jewish immigrants from Yemen, who settled there in December 1949. They worked in land reclamation and forestry. The Jewish National Fund established a 45-dunam nursery in Eshtaol to supply saplings for JNF forests. Later, the moshav branched out into poultry and other agricultural enterprises. At the end of the 1990s, the moshav absorbed 100 new families.
Eshtaol was in the territory allotted to the tribe of Dan (Joshua 19:41). According to the Bible, Samson began to prophesy "between Zorah and Eshtaol" (Book of Judges 13:25). After his death in Gaza, Samson's body was brought back for burial in his father’s tomb between Eshtaol and Zorah (Judg 16:31). Five scouts from Eshtaol and Zorah were sent out to find a land suitable for the tribe of Dan. (Judges 18:1-29).