Purpose: The aim of this study was to review the available literature and define clinical practice guidelines for the use of agents for the prevention and treatment of gastrointestinal mucositis. Methods: A systematic review was conducted by the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer/International Society of Oral Oncology (MASCC/ISOO). The body of evidence for each intervention, in each cancer treatment setting, was assigned an evidence level. Based on the evidence level, one of the following three guideline determinations was possible: recommendation, suggestion, and no guideline possible. Results: A total of 251 clinical studies across 29 interventions were examined. Panel members were able to make one new evidence-based negative recommendation; two new evidence-based suggestions, and one evidence-based change from previous guidelines. Firstly, the panel recommends against the use of misoprostol suppositories for the prevention of acute radiation-induced proctitis. Secondly, the panel suggests probiotic treatment containing Lactobacillus spp., may be beneficial for prevention of chemotherapy and radiotherapy-induced diarrhea in patients with malignancies of the pelvic region. Thirdly, the panel suggests the use of hyperbaric oxygen as an effective means in treating radiation-induced proctitis. Finally, new evidence has emerged which is in conflict with our previous guideline surrounding the use of systemic glutamine, meaning that the panel is unable to form a guideline. No guideline was possible for any other agent, due to inadequate and/or conflicting evidence. Conclusions: This updated review of the literature has allowed new recommendations and suggestions for clinical practice to be reached. This highlights the importance of regular updates.