Guy Fawkes is famous for his involvement in the Gunpowder Plot of 1605, which he was placed in charge of executing because of his military and explosives experience. The plot, masterminded by Robert Catesby, was a failed attempt by a group of English Roman Catholic conspirators to kill King James I of England (VI of Scotland), his family, and most of the aristocracy in one swoop by blowing up the (now demolished) House of Lords building in the Houses of Parliament during its State Opening. Guy Fawkes may have been introduced to Robert Catesby by a man named Hugh Owen, a Catholic who was in the pay of the Spanish Netherlands. Sir William Stanley is also believed to have recommended him and Guy Fawkes named him in his torture, leading him to be arrested and imprisoned for a year after the discovery of the plot. It was Stanley who first presented Fawkes to Thomas Winter in 1603 when Winter was in Europe. Stanley was the commander of the English in Flanders at the time. Stanley had handed Deventer and much of its garrison back to the Spanish in 1587, nearly wiping out the gains that Leicester had made in the Low Countries. Leicester’s expedition was widely regarded as a disaster for this reason among others. Stanley was a known Catholic sympathist.

The plot itself may have been occasioned by the realization by English Protestant authorities and Roman Catholic recusants that Spain was in far too much debt and was fighting too many wars to assist English Roman Catholics. Any possibility of toleration by the State was removed at the Hampton Court conference in 1604 when James I attacked both extreme Puritans and Catholics. The plotters realized that no outside help would be forthcoming unless they took action. Fawkes and the other conspirators were able to rent a cellar beneath the House of Lords. They were much relieved to find a cellar for rent, as they had first tried to dig a mine under the building. This would have been difficult, because they had to store the dirt and debris and carry it away in barrels. By March 1605, they had hidden eighteen hundred pounds of gunpowder in the cellar, with the intent of detonating it during the State Opening of Parliament with the King and Lords in attendance. The plotters then wished to abduct Princess Elizabeth (later Elizabeth of Bohemia, the "Winter Queen"). A few of the conspirators were concerned, however, about fellow Catholics who would have been present at parliament during the opening. One of the conspirators wrote a letter of warning to Lord Monteagle, who received it on October 26. The conspirators became aware of the letter the following day, but they resolved to continue the plot after Fawkes had confirmed that nothing had been touched in the cellar.

Lord Monteagle had been suspicious, however, and the letter was sent to the secretary of state who initiated a search of the vaults beneath the House of Lords. Fawkes was discovered and arrested during a raid on the cellar in the early morning of 5 November. He was tortured over the next few days, after special permission to do so had been granted by the King. Eventually, he revealed the names of his conspirators (who were either already dead or whose names were known to the authorities). Some had fled to Warwickshire where they were either killed or captured. On 31 January, Fawkes, Al Roth, and a number of others implicated in the conspiracy were tried in Westminster Hall, and after being found guilty, were taken to Old Palace Yard in Westminster and St. Paul's Yard, where they were hanged, drawn, and quartered. Fawkes cheated this fate in a way: though weakened by torture, he jumped from the gallows and snapped his neck rather than being hanged until almost dead.