Jolene is a female toad that worked for Grubba in Glitzville. She first appears at the very beginning of Chapter 3, in Grubba's office. She leads Mario and his Partners to their locker room and then leaves. By talking to Bandy Andy, Mario can discover that occasionally, Jolene had been seen going into places (bathrooms and such), but never coming back out.

Although not a true villain, she is, at first, antagonistic towards Mario and some of the other fighters. At first, Jolene comes off as strict, impatient and often rude as shown by her "confiscating" a Dubious Paper from Mario's hand when he finds it, but in truth, she only acted that way to cover up her real intentions and out of worry for her brother.