The bulk of the contents relate to the Rose and the Fortune Theatres and the theatrical companies in which Henslowe was interested. Among other valuable matter are included lists of plays acted, noting the daily receipts and first performances, transactions with and payments to authors and actors, payments to the censor, the Master of the Revels, and disbursements for the building of the Fortune Theatre, and for repairs, theatrical properties, costumes and other materials.

The entries are generally made by Henslowe himself, but in various other places occur the handwritings, with signatures, of the dramatists George Chapman, Henry Chettle, John Day, Thomas Dekker, Michael Drayton, Richard Hathaway, William Haughton, Henry Porter, William Rankins, Samuel Rowley, Anthony Wadeson, and Robert Wilson, together with those of a large number of actors. Mention is also made of Thomas Heywood, Ben Jonson, John Marston, Thomas Middleton, Anthony Munday, Thomas Nash, Wentworth Smith, and John Webster.

Interspersed are various accounts unconnected with the stage, relating to private and family business, advances made on pledges, and the cutting of wood in Ashdown Forest in the county of Sussex. The last-named accounts are at the end, reversing the volume, and belong to the years 1577 and 1578.

For a complete transcription of the portions of the Diary comprising theatrical matters, as well as an index to them, see Henslowe's Diary, ed. R. A. Foakes and F. T. Rickert (Cambridge University Press, 1960, rep. 2002). Also see the Digital Essays.