The chemotherapeutic agent 5-Fluorouracil (5-FU) can induce salivary gland hypofunction (SGH); however, previous studies did not reach final conclusions on the influence of this drug on glandular tissue. Thus, the aim of this study was to investigate the effect of 5-FU on submandibular (SMs) and sublingual glands (SLs), as well as, the effect of laser phototherapy (LPT) on SGH induced by 5-FU. Eighty-five hamsters were divided into three groups: control (C), chemotherapy (CT) and laser (L), and the SGH was induced by two injections of 5-FU in groups CT and L. The irradiation was performed using a diode (lambda 780nm/20mW/5Jcm(-2)/0.2J and 10s per point/spot size of 0.04cm(2)) and applied daily. On the euthanasia day, SMs and SLs were removed and biochemical analyses were carried out. The lactate dehydrogenase activity was increased in group CT when compared with group C for SLs and SMs (P<0.05). In addition, the peroxidase and catalase activities were increased and superoxide dismutase was decreased by 5-FU (P<0.05). However, LPT appears to be a protective mechanism against oxidative stress, tending to alter the activity of these antioxidant enzymes, suggesting LPT as a promising therapy to modulate the 5-FU harmful effect.