The Civil War affected Sculcoates because of its location, next to Hull. Throughout the war, Hull supported the Parliamentarian cause but initially, the surrounding area was in Royalist hands.  In 1642 an attempt was made to take the town and secure the arsenal in it when Royalist forces besieged Hull. During the course of the siege, men from Hull's garrison slipped out of the town and cut the river banks, flooding the surrounding area. The effect on Sculcoates was disastrous. Great efforts had always been taken to maintain the banks and this deliberate flooding caused much damage and ruined the harvest.

1643 saw the second siege of Hull when Royalists in Cottingham and Newland began to pour red hot shot over Sculcoates onto the town in an attempt to start fires. Fairfax, the Governor of Hull, ordered the destruction of the remaining Charterhouse buildings as they were too close to the town walls and it was feared that they would give cover to the town's enemies. The inhabitants of the Charterhouse Hospital were evacuated to a house in Whitefriargate for the duration and artillery was positioned on the site of the Charterhouse to bombard the Royalist guns. Later the sluices were opened, causing widespread flooding, and for the second year in succession the Sculcoates harvest was lost. Eventually the siege was lifted and fighting switched to other parts of the country.

1649, after the war, the Hospital was rebuilt on the old site, on the south side of Charterhouse Lane. In 1663 a new building was erected on the north side and in 1673 a new chapel was added.