NontyphoidalSalmonellaspecies are one of the main bacterial causes of foodborne diseases, causing a public health problem. In addition, the ability to form biofilms, multiresistance to traditional drugs, and the absence of effective therapies against these microorganisms are some of the principal reasons for the increase in bacterial diseases. In this study, the anti-biofilm activity of twenty essential oils (EOs) onSalmonella entericaserovar Enteritidis ATCC 13076 was evaluated, as well as the metabolic changes caused byLippia origanoidesthymol chemotype EO (LOT-II) on planktonic and sessile cells. The anti-biofilm effect was evaluated by the crystal violet staining method, and cell viability was evaluated through the XTT method. The effect of EOs was observed by scanning electron microscopy (SEM) analysis. Untargeted metabolomics analyses were conducted to determine the effect of LOT-II EO on the cellular metabolome. LOT-II EO inhibitedS. Enteritidis biofilm formation by more than 60%, without decreasing metabolic activity. Metabolic profile analysis identified changes in the modulation of metabolites in planktonic and sessile cells after LOT-II EO treatment. These changes showed alterations in different metabolic pathways, mainly in central carbon metabolism and nucleotide and amino acid metabolism. Finally, the possible mechanism of action ofL. origanoidesEO is proposed based on a metabolomics approach. Further studies are required to advance at the molecular level on the cellular targets affected by EOs, which are promising natural products for developing new therapeutic agents againstSalmonellasp. strains.