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<i>Thymus vulgaris</i> essential oil has potential good biological activity. The aim of the research was to evaluate the biological activity of the <i>T. vulgaris</i> essential oil from the Slovak company. The main components of <i>T. vulgaris</i> essential oil were thymol (48.1%), <i>p</i>-cymene (11.7%), 1,8-cineole (6.7), γ-terpinene (6.1%), and carvacrol (5.5%). The antioxidant activity was 85.2 ± 0.2%, which corresponds to 479.34 ± 1.1 TEAC. The antimicrobial activity was moderate or very strong with inhibition zones from 9.89 to 22.44 mm. The lowest values of MIC were determined against <i>B. subtilis</i>, <i>E. faecalis,</i> and <i>S. aureus</i>. In situ antifungal analysis on bread shows that the vapor phase of <i>T. vulgaris</i> essential oil can inhibit the growth of the microscopic filamentous fungi of the genus <i>Penicillium</i>. The antimicrobial activity against <i>S. marcescens</i> showed 46.78–87.80% inhibition at concentrations 62.5–500 µL/mL. The MALDI TOF MS analyses suggest changes in the protein profile of biofilm forming bacteria <i>P. fluorescens</i> and <i>S. enteritidis</i> after the fifth and the ninth day, respectively. Due to the properties of the <i>T. vulgaris</i> essential oil, it can be used in the food industry as a natural supplement to extend the shelf life of the foods.