[2] He held administrative offices in Lincolnshire, and was one of the commissioners who tried Philip Howard, 20th Earl of Arundel, for treason on 14 April 1589.

Willoughby's second son, Ambrose, was one of the Queen's esquires of the body, and in early 1598 was involved in a brawl with Henry Wriothesley, 3rd Earl of Southampton.

Ambrose Willoughby had ordered Southampton to leave the presence chamber where he was playing at primero after the Queen had retired for the evening.

[8] There is a suggestion that underlying the altercation was something Willoughby had said which caused trouble between Southampton and his mistress, Elizabeth Vernon, one of the Queen's Maids of Honour.

[8] Ambrose Willoughby is also mentioned in a letter of 17 June 1602 from John Chamberlain to Sir Dudley Carleton: "Gray Bridges hath hurt Ambrose Willoughby in the heade and body, for abusing his father and himself at a conference of arbiterment twixt them and Mistris Bridges".