Croton is the second largest genus of the family Euphorbiaceae. The species C. antisyphiliticus Mart., from Cerrado biome, is popularly known as pé-de-perdiz and widely used in folk medicine due to its therapeutic properties. The aqueous root extract of this species is popularly used for sexually transmitted infections, diseases of the reproductive system, colic treatment, rheumatism, against fungi, ulcer wounds and inflammation. In this work, an ethnobotanical study was carried out in the Coqueiros community, located in Catalão, Goiás state, and C. antisyphiliticus was mentioned to antibiotic, infection of the uterus and influenza. This information instigated the chemical and anti-inflammatory studies of the ethanolic and aqueous extracts from roots to verify and better elucidate therapeutic effects of these. The roots of C. antisyphiliticus were identified and collected; the testimony species is deposited in the Herbarium of the Federal University of Goiás, under the code UFG-27,850, as well as registered in the SisGen under the code A11AE20. Preparation of crude extracts of the roots occurred by two methodologies: 1. following the Coqueiros community method boiling the fresh roots in water and freeze-drying the solvent, obtaining the aqueous root extract (EAR); 2. extraction of the dried roots with ethanol for nine days (exchange every three days), followed by evaporation of the solvent in rotary evaporator, low pressure, obtaining the ethanolic extract of the roots (EER). The chemical characterization of extracts was performed by liquid chromatography–mass spectrometry (LC-MS) using a Thermo Scientific Q Exactive Focus LC-MS/MS equipment coupled to a UHPLC Dionex UltiMate 3000 and data were analyzed using GNPS platform and Cytoscape software to obtain molecular networks. The antinociceptive activity of extracts was evaluated in the Irwin test, acetic acid-induced writhing test, formalin-induced pain test and tail flick test. The chemical characterization revealed the presence of phenolic compounds in both the extracts. Gallic acid, protocatechuic acid, catechin, epicatechin, gentisic acid, luteolin and kaempferol were identified in the EER; and epicatechin, gentisic acid and caffeic acid in EAR. In addition, by the dereplication using GNPS platform was identified L-gulonolactone in EER and also gallic acid, protocatechuic acid, ellagic acid and catechin compounds. In Irwin test, EER [doses of 125, 250 and 500 mg kg-1 (p.o.)] and EAR [doses of 300, 600, 900, 1200 or 1500 mg kg-1 (p.o.)] were determined to be used in subsequent pharmacological tests. The treatment with EER [125, 250 and 500 mg kg-1 (p.o.)] reduced the number of writhing in a dose-dependent manner by 30, 53 and 41%, respectively, when compared to the vehicle group. In formalin-induced pain, EER [250 mg kg-1 (p.o.)] decreased the licking time in the first and second phase by 36 and 31%, respectively, when compared to the vehicle group. However, this same dose of EER did not cause effect in tail flick test. Treatment with EAR at doses and 600, 900, 1200 or 1500 mg kg-1 (p.o.) reduced the number of writhing by 14, 25, 44 and 44%, respectively, compared to the vehicle group. These results showed significant antinociceptive effect of EER and EAR, which may be associate to the presence of phenolic compounds identified in both the extracts, since the antinociceptive and anti-inflammatory activities of these constituents are described in the literature. In conclusion, the present study suggests an antinociceptive activity of EER and EAR from C. antisyphiliticus, which may be association to presence of phenolic compounds. However, further studies are needed to investigate the mechanisms of action involved in this effect.