She was born in Rome to a wealthy and prominent family. Though her father Pretexatus was a pagan, her mother Fausta instructed her in the things of God. Her father married her against her will to Publius, a prodigal and impious man lacking in Christian or pagan virtue. Anastasia was in the custom of dressing herself as a poor working woman and going out by night to visit and comfort the many Christians in prison (this was the time of Diocletian's persecution). When Publius discovered this, he was furious that his wife was demeaning herself by consorting with the despised Christians, and had his wife locked in the house with so little food that she came close to death by starvation. She was able to get a letter to her spiritual father Chrysogonus, who was also in prison, and their correspondence helped to sustain her through her ordeal. After three months her husband died in a shipwreck and she regained her freedom. Immediately she redoubled her work for the suffering Christians and their families, devoting all her time and wealth to their comfort and care.

One day Diocletian declared that all Christians in his prisons should be slain, and his command was carried out in one night. The next day Anastasia came to visit her beloved companions and, learning that all were dead, fell sobbing by the gate, no longer caring to conceal her Christian faith from anyone. Almost immediately she was arrested and brought before the authorities, who subjected her to every form of abuse. One prefect offered to marry her if she would bow to the idols, but to have her tortured to death if she would not. When she was unmoved, he attempted to rape her, but was struck blind and died miserably. She then briefly escaped to Nicaea and found refuge with the pious St Theodota, but was seized again along with Theodota and her children. After further trials and torments Anastasia, Theodota and her children, and others who had been converted to Christ through Anastasia's example, were executed.

Saint Anastasia's relics were taken to Rome, where a church was built in her honor. The relics were later translated to Constantinople and placed in another church bearing her name, where they worked many miracles. Because she has healed many through her prayers from the effects of poisons and potions, she is called Pharmocolytria, "Deliverer from Potions."