Anthony Babington was descended from a family of great antiquity who in successive generations had acquired vast estates in and around Derbyshire. He was born the third son of Henry Babington, and his second wife Mary, daughter of George, Lord Darcy, and granddaughter of Thomas, Lord Darcy, who was executed in 1538 as a principal conspirator in the "Pilgrimage of Grace". His father died in 1571 when he was nine, and his mother remarried to Henry Foljambe. Under his three guardians (his mother, Foljambe and Philip Draycot of Paynsley, Staffordshire), Babington was indebted for his education.

Although all three outwardly conformed to Protestantism, it is certain they were all church papists. Even Babington's father, while he was alive, was said to have been "inclined to papistrie", and had a brother who was "a doctor of divinitie of the same religious profession". The Foljambes were renowned papists in their own right, and many suffered for their faith. Babington apparently remained in Dethick until about 1577, when he was briefly a page to Mary Queen of Scots while she was under the care of the Earl of Shrewsbury. It is during these brief few months that many claim Babington became utterly devoted to Mary and her cause to sit on the throne of England. In 1579 he married Margery Draycot, and by 1580 was in London studying law.

Soon after his admission to Lincoln's Inn, Babington abandoned the bar for fashionable town life. His wealth, charm and good looks soon secured him a large following around court, and it was inevitable that other Catholics, seduced by Jesuit stirrings abroad, soon formed his inner circle of friends. Early in 1580, Babington was one of a secret circle established for the protection of priests, most notably Edmund Campion and Robert Parsons. With the fundamental basis of the protection and maintenance of Jesuit missionaries, the group were soon being commended in private by Pope Gregory XIII. Babington briefly visited the continent at this time, spending six months in France where it is believed he may have first met some of Elizabeth's enemies.

Babington was thus drawn into the plot that generally bares his name - a plot hatched by Mary's supporters in France to assassinate Elizabeth and place Mary on the throne. Babington's correspondence with her eventually led to him revealing the details of the plot sealing everyone's fate, including Mary's.

On 13-14 September, Babington, Ballard and five others (the poet Chidiock Tichbourne, Thomas Salisbury, Robert Barnewell, John Savage and Henry Donn) were placed on trial. Babington confessed all, but placed all the blame on Ballard, who graciously admitted that he wished the spilling of his blood could save his young friend. Two days later, seven more conspirators (Edward Habington, Charles Tilney, Edward Jones, John Charnock, John Travers, Jerome Bellamy, and Robert Gage) were similarly tried and sentenced to be hung, drawn and quartered.

On 19 September, Babington wrote to Elizabeth begging her to employ mercy and spare him. On the same day, he offered a friend 1000li if he could secure his release. The following day, the first seven were drawn on hurdles from Tower Hill to St Giles. Ballard suffered at the hands of the executioner first, undergoing terrible torture before his life was extinguished. Babington followed and suffered a similarly barbaric execution, being still alive as the executioners knife went to work on disemboweling him. Elizabeth was horrified at the revolting cruelty of their death, and ordered that those to be executed the following day were to be left hanging until dead before being cut down. And so it was that on 21 September, the remaining seven conspirators were put to death.

By his wife Margery, Anthony Babington had a daughter, who died at the age of eight, probably before her father.