Mitracarpus hirtus (L.) DC. (Family: Rubiaceae) is a tropical annual herb commonly found in America and Mexico. In the present study, the methanol, ethyl acetate, dichloromethane and aqueous extracts of the plant were tested for total phenolic (TPC) and flavonoid content (TFC) and antioxidant activities were evaluated using a battery of assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity, total antioxidant capacity (TAC) (phosphomolybdenum) and metal chelating. Enzyme inhibitory effects were investigated using acetylcholinesterase (AChE), tyrosinase, alpha-amylase and alpha-glucosidase as target enzymes. The phytochemical profile was obtained using liquid chromatography-diode array detection-electrospray ionization-mass spectrometry (LC-DAD-ESI-MS(n)), liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS(n)) and nuclear magnetic resonance (NMR) experiments. Results showed that the dichloromethane and ethyl acetate extracts yielded the highest TPC (29.10 +/- 0.07 mg gallic acid equivalent/g) and TFC (38.14 +/- 0.91 mg rutin equivalent/g), respectively. Aqueous extract showed weak activity against tested enzymes but demonstrated the strongest ABTS scavenging activity (59.39 +/- 1.19 mg trolox equivalent/g) and is the strongest Fe(3+) reducer (59.42 +/- 0.59 mg trolox equivalent/g). Phytochemical analysis revealed the presence of phenolics, pyrrolizidine alkaloids and triterpene acid. This is the first report gathering scientific data on antioxidant, enzyme inhibitory activities and phytochemical composition of M. hirtus and the obtained results can be used as starting point for further investigation on this traditional medicinal herb. CI - Copyright (c) 2020 Elsevier B.V. All rights reserved.