The community was initially formed as an urban kibbutz in Givat Michael near Ness Ziona by immigrants from Poland (who were members of the "BaMesilah" group from which the kibbutz takes its name) and Bulgaria (members of the "Bulgaria - Tel Hai group"), with both groups belonging to the Hashomer Hatzair movement.
The kibbutz itself was established on 22 December 1938 as a tower and stockade settlement. During the 1948 Arab-Israeli War, the kibbutz was bombed by an Iraqi aeroplane. One member of the kibbutz was killed in the bombing, and some other members were injured.
As well as agriculture, Mesilot has a large and successful factory, the "Mesilot Cable Factory." Likewise, the kibbutz does various forms of agriculture, including livestock, a fishery, an orchard, plantings of olives and dates, and other vegetables.
The children of the kibbutz are educated in the regional school within the kibbutz, which also serves children from the nearby kibbutzim Reshafim, Nir David, and Beit Alfa. Next to the elementary school, based on the donation of Mrs. Yehudit ben Moshe (who was a member of Mesilot and the first principal of its school), a special school was founded for children with autism. Normal children from grades 7 through 12 learn in the Gilboa School, named for the heroes of the Warsaw Ghetto uprising, which was founded next to the Beit Alfa kibbutz and is used jointly by Mesilot, Reshafim, Nir David and Beit Alfa. In 2005 this school was closed, so the students of Mesilot transferred to the regional school in Neve Eitan.