The aim of this study was to evaluate the antinociceptive effect of Kaempferol-3-O-rutinoside (KR), isolated from the plant Ouratea fieldingiana, on the orofacial nociception and possible mechanisms of action. Adult zebrafish (Danio rerio) were tested as a behavioral model to study formalin, glutamate, capsaicin, cinnamaldehyde and acidic saline-induced orofacial nociception, using as parameter the number of times the fish crossed the lines between the quadrants of a glass Petri dish during a specific time. Morphine was used as positive control. The effect of KR was tested for modulation by opioid (naloxone), nitrergic (L-NAME), TRPV1 (ruthenium red), TRPA1 (camphor) or ASIC (amiloride) antagonists. The effect of KR on zebrafish locomotor behavior was evaluated with the open field test. KR did not alter the fish’s locomotor system and significantly reduced the orofacial nociceptive behavior induced by all noxious agents compared to the control group. The antinociceptive effect of KR was similar to morphine. All antagonists inhibited the antinociceptive effect of KR. KR has pharmacological potential for the treatment of acute orofacial pain and this effect is modulated by the opioid and nitrergic systems as well as TRPV1, TRPA1 and ASIC channels. These results can lead to the development of a new natural product for the treatment of orofacial pain and confirm the popular use of O. fieldingiana leaf for pain relief.