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The aim of this study was to analyze the chemical composition and biological and antibiofilm activity of the essential oil (EO) of <i>Thymus serpyllum</i> with the use of a MALDI-TOF MS Biotyper. The main compounds of the EO were thymol, 18.8%; carvacrol, 17.4%; o-cymene, 15.4%; and geraniol, 10.7%. It was found that free-radical scavenging activity was high. The highest antimicrobial activity was observed against <i>Pseudomonas aeruginosa</i>, <i>Salmonella enteritidis,</i> and biofilm-forming bacteria. The changes in the biofilm structure after <i>T. serpyllum</i> EO application confirmed the inhibitory action and the most pronounced effect was observed on <i>Bacillus subtilis</i> biofilm. The antifungal activity of the vapor phase was the most effective against <i>Penicillium crustosum. T. serpyllum</i> should be a suitable alternative to synthetic antioxidants as well as antimicrobials. The EO of <i>T. serpyllum</i> can be used in the vapor phase in the storage of root vegetables as well as a growth inhibitor of <i>Penicillium</i> on bread.