Among Eastern Orthodox and Eastern Catholic Christians, holy water is blessed in the church and given to the faithful to drink at home when needed and to bless their homes. In the weeks following the Feast of Epiphany, clergy visit the homes of parishioners and conduct a service of blessing by using the holy water that was blessed on the Feast of Theophany. For baptism, the water is sanctified with a special blessing.

Throughout the centuries, there have been many springs of water that have been believed by members of the Orthodox Church to be miraculous. Some still flow to this day, such as the one at Pochaev Lavra in Ukraine, and the Life-Giving Spring of the Theotokos in Constantinople (commemorated annually with the blessing of holy water on Bright Friday).

Although Eastern Orthodox do not normally bless themselves with holy water upon entering a church like Catholics do, a quantity of holy water is typically kept in a font placed in the narthex (entrance) of the church, where it is available for anyone who would like to take some of it home with them. It is customary for Orthodox to drink holy water, to use it in their cooking and to sprinkle their houses with it.

Often, when objects are blessed in the church (such as the palms on Palm Sunday, Icons or sacred vessels) the blessing is completed by a triple sprinkling with holy water using the words, "This (name of item) is blessed by the sprinkling of this holy water, in the name of the Father, and of the Son, and of the Holy Spirit."

Holy water is sometimes sprinkled on items or people when they are blessed outside the church building, as part of the prayers of blessing. In Russia, it is common for Orthodox Christians to bring newly bought cars to the church for blessing. Holy water is sprinkled inside and out, as well as under the hood. Similarly, in Alaska, the fishing boats are sprinkled with holy water at the start of the fishing season as the priest prays for the crews' safety and success. Some Catholics also have a priest bless their cars or homes with holy water as a way of invoking God's blessing and protection.

Orthodox Christians most often bless themselves with holy water by drinking it. It is traditional to keep a quantity of it at home, and many Orthodox Christians will drink a small amount daily with their morning prayers. It may also be used for informal blessings when no clergy are present. For example, parents might bless their children with holy water before they leave the house for school or play. It is also often taken with prayer in times of distress or temptation.

There are two rites for blessing holy water: the Great Blessing of Waters which is held on the Feast of Theophany, and the Lesser Blessing of Waters which is conducted according to need during the rest of the year. Both forms are based upon the Rite of Baptism. Certain feast days call for the blessing of Holy Water as part of their liturgical observance.

The use of holy water is based on the Baptism of Jesus by John the Baptist in the River Jordan, and the Orthodox interpretation of this event. In their view, John's baptism was a baptism of repentance, and the people came to have their sins washed away by the water. Since Jesus had no sin, but was God incarnate, his baptism had the effect not of washing away Jesus' sins, but of blessing the water, making it holy—and with it all of creation, so that it may be used fully for its original created purpose to be an instrument of life.