The genusThymusL., belonging to the Lamiaceae family, contains about 220 species with a distribution that mainly extends in Europe, northwest Africa, Ethiopia, Asia, and southern Greenland. Due to their excellent biological properties, fresh and/or dried leaves and aerial parts of severalThymusssp. have been utilized in the traditional medicine of many countries. To evaluate not only the chemical aspects but also the biological properties, the essential oils (EOs), obtained from the pre-flowering and flowering aerial parts ofThymus richardiisubsp.nitidus(Guss.) Jalas, endemic to Marettimo Island (Sicily, Italy), were investigated. The chemical composition of the EOs, obtained by classical hydrodistillation and GC-MS and GC-FID analyses, showed the occurrence of similar amounts of monoterpene hydrocarbons, oxygenated monoterpenes, and sesquiterpene hydrocarbons. The main constituents of the pre-flowering oil wereβ-bisabolene (28.54%),p-cymene (24.45%), and thymol methyl ether (15.90%). The EO obtained from the flowering aerial parts showed as principal metabolitesβ-bisabolene (17.91%), thymol (16.26%), and limonene (15.59%). The EO of the flowering aerial parts, and its main pure constituents,β-bisabolene, thymol, limonene,p-cymene, and thymol methyl ether were investigated for their antimicrobial activity against oral pathogens and for their antibiofilm and antioxidant properties.