Warden Gilbert Sheldon was the leader of a strongly Royalist College whose Fellows were to be put to the test by the events of the Civil War and the Commonwealth. Almost all of them contributed generously to the Royalist cause and sacrificed their fellowships rather than swear allegiance to Parliament. The College was second only to Magdalen in the amount of plate (over 250 lb) that it supplied to the Royal Mint, and it lent the king a further £654 14s 3d – a bad debt not finally written off until 1857. In spite of failure of rents, it provided maintenance for Royalist soldiers and contributed towards fortifications and stores. Small wonder that Sheldon was forcibly removed from the lodgings in April 1648 and that only seven of the forty Fellows avoided the loss of their fellowships. John Palmer was brought in to take the place of Warden Sheldon, and between 1648 and 1652 forty-three men (thirteen of them from Cambridge) were intruded into fellowships by the Commissioners. Most of the intruders were academically undistinguished, but they included Thomas Sydenham – one of a number of medical and scientific Fellows of the college in the second half of the century, some of whom were early recruits to the ranks of the Royal Society. In 1653, when regular elections commenced, Christopher Wren joined All Souls after three years at Wadham. He held College office and in 1658 (the year of his bursarship) designed the fine and accurate sun-dial which surmounted the south wall of the chapel until its removal to the Great Quadrangle in 1877.

The Restoration of King Charles II in May 1660 brought welcome relief to the College. All the intruded Fellows save one (Edward Greene) were sent packing, Sheldon was reinstated as Warden, and the few surviving pre-Civil War Fellows who wished to resume their fellowships rejoined the College. The chapel received attention in the early 1660s: a screen based on a design by Wren was installed, a Last Judgement fresco was painted over the east end and painted wooden panels were inserted between the roof-trusses in the chancel. These paintings were the work of Isaac Fuller, whose fresco was to justify John Evelyn's comment that it would not 'hold long', being 'too full of nakeds'. Parts only of some of the painted panels have defied prophecy and are preserved in the antechapel.

In 1681 Warden Jeames, supported by the Visitor, Archbishop Sancroft, at last succeeded by the use of his veto in stamping out the abuse of corrupt resignations, and the quality of the fellowship notably improved when men such as George Clarke (Fellow, 1680), Nathaniel Lloyd (1689) and Christopher Codrington (1690) joined the College. Jeames's successor as Warden, however, represented a falling-off. This was the Hon. Leopold Finch, a Fellow of only five years' standing in the College when in January 1687 he was, at the age of twenty-four, unconstitutionally nominated to the wardenship by King James II (he was not formally admitted Warden until 1698). A man of some learning, with a passion for sash windows and with low moral standards, he died in the lodgings (which he was sharing with bailiffs) in November 1702.