The aim of this study was to determine the chemical profile of Thymus nummularius by LC-MS/MS and GC/MS. Additionally, the methanol extract, essential oil and some individual main compounds of Thymus nummularius were tested for antioxidant, anticholinesterase and antimicrobial activities. Besides, HPLC-FLD was used to determine total aflatoxin in the plant material. Among the twenty-seven compounds studied by LC-MS/MS, rosmarinic acid (131,898.9 +/- 6463.0 mu g/g dry-extract) was found to be the most abundant compound in the methanol extract. On the other hand, thymol (60.38%) and terpinyl-acetate (10.49%) were found to be the major compounds of the essential oil. Both the essential oil and the methanol extract of T. nummularius exhibited strong antioxidant activity in the four tested methods. Furthermore, the essential oil showed high inhibitory activity against acetyl-,butyryl-cholinesterase enzymes and very strong antimicrobial activity against all tested microorganisms. Besides, T. nummularius can be used both as rosmarinic acid source and as food supplement due to its non-aflatoxin content and high antioxidant, anticholinesterase and antimicrobial activities. (C) 2015 Elsevier B.V. All rights reserved.