Aside from those who reside in the playhouse, the show featured numerous guests. These people would drop in for various reasons, from mere friendship to practical matters. While there, these visitors to the playhouse would usually interact with Pee-Wee and his many gadgets and creatures.
- Captain Carl - gruff sailor with a very low tolerance level
- Cowboy Curtis - prototypical Wild West cowboy
- Dixie - taxi driver for the King of Cartoons; only appeared during Season 1
- King of Cartoons - showed old-fashioned cartoons
- Miss Yvonne - Pee-wee's friend and love interest with an obsession for beauty and fashion; also known as "The Most Beautiful Woman in Puppetland"
- Mrs. Rene - replaced Mrs. Steve as Pee-Wee's neighbor
- Mrs. Steve - chronic "snooper" in the playhouse with an apparent lack of charm and friendliness; only appeared during Season 1
- Pee-Wee Herman - star of the show; owned the playhouse
- Playhouse Gang - group of kids who stopped by on occasion to play various games
- Reba the Mail Lady - delivered Pee-Wee's mail; is notably a replacement character for Mailman Mike from The Pee-wee Herman Show
- Ricardo - replaced Tito; had an apparent background in medicine and soccer
- Tito - Latino lifeguard for the playhouse pool; only appeared during Season 1
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