Patients undergoing hematopoietic stem cell transplantation (HSCT) are submitted to a conditioning regimen of high-dose chemotherapy, with or without radiation therapy, which usually results in oral ulcerations and mucosal barrier breakdown. Oral mucositis (OM) is a common and debilitating toxicity side effect of autologous and allogeneic HSCT. The aim of this study was to evaluate the effect of low-level laser therapy (LLLT) on the severity of OM and inflammatory mediator (TNF-α, IL-6, IL-1β, IL-10, TGF-β, metalloproteinases, and growth factors) levels in saliva and blood of HSCT patients. Thirty patients were randomly assigned to two groups: control (n = 15) and laser (n = 15). LLLT was applied from the first day of the conditioning regimen until day 7 post-HSCT (D + 7). Saliva and blood were collected from patients on admission (AD), D-1, D + 3, D + 7, and on marrow engraftment day (ME). Clinical results showed less severe OM in the laser group (p < 0.05). The LLLT group showed increased matrix metalloproteinase 2 (MMP-2) levels in saliva on D + 7 (p = 0.04). Significant differences were also observed for IL-10 on D + 7 and on ME in blood plasma, when compared to the control group (p < 0.05). No significant differences were seen in saliva or blood for the other inflammatory mediators investigated. LLLT was clinically effective in reducing the severity of chemotherapy-induced OM in HSCT patients, and its mechanism of action does not seem to be completely linked to the modulation of pro- or anti-inflammatory cytokines, growth factors or matrix metalloproteinases.