In the present investigation, we determined the in vitro antimicrobial activity of eight essential oils (EOs) and three medium-chain fatty acids (MCFAs) alone and in combination against Staphylococcus aureus ATCC 700698, Klebsiella pneumoniae ATCC 700603, and E. coli FcW5. The interactions between EOs and MCFAs were determined by fractional inhibitory concentration indices. Moreover, mode of action of selected bioactive components was studied by changes in bacterial surface charge, morphology, and membrane integrity assays. Among EOs, carvacrol (CAR), trans-cinnamaldehyde (TC), and thymol (TM) showed strong antimicrobial activity. In combination study, CAR+OA (octanoic acid), CAR+DA (decanoic acid), and TM+OA were observed as the most significant (P≤0.05) which were also confirmed through time-kill plots. Based on these results, CAR+OA were found to be most efficacious in terms of killing time (P≤0.05). Changes in the surface charge, morphology, and membrane integrity upon the combined treatment of CAR+OA were also observed, which ultimately leads to cell death. Results suggest that CAR+OA when used in combination offer a significant (P≤0.05) additive antimicrobial activity against the selected pathogenic bacteria. Therefore, these natural bioactive molecules could be interesting alternatives to conventional therapy for the control of mastitis caused by multi-drug-resistant pathogens in bovine animals to ensure the milk safety.