Bovine mastitis has become a significant economic importance for dairy industry. Concerns regarding poor milk quality, and emergence of bacterial resistance has necessitated to develop alternative therapeutic approach to antibiotics for treatment of mastitis. Saturated medium-chain fatty acids (MCFAs) and essential oils (EOs) are known natural antimicrobials, but their combined effect has not been investigated extensively. The objective of the present investigation was to examine the bactericidal effect of various combined treatments of eight EOs and three saturated MCFAs to inactivate predominant mastitis pathogens, including Staphylococcus aureus ATCC 29213; Escherichia coli ATCC 25922; Klebsiella pneumoniae ATCC 27736; and Streptococcus agalactiae ATCC 27956. The minimum inhibition concentration (MIC) values confirmed that all the tested pathogens were variably susceptible to both EOs and saturated MCFAs. Among essential oils, carvacrol (CAR), trans-cinnamaldehyde (TC), and thymol (TM) showed highest inhibitory activity at concentration 0.38-1.32 mg/mL. Carvacrol exhibited effective additive antibacterial activity in combined treatment with octanoic acid (OA) in terms of its fractional inhibitory index (0.63-0.88) and time-kill effect in reducing about 6 log CFU/mL bacterial cells in less than 5 min. Effort was also made to elucidate mechanism of antibacterial action of CAR and OA against selected mastitis pathogens by observing changes in cell microstructure, permeability and integrity of cell membrane and their membrane potential. After adding CAR and OA at MIC level, there were obvious changes in cell morphology, leakage of small electrolytes, and macromolecules at the initial few hours of treatment i.e. within 1-2 h were observed. Our results indicated that CAR and OA could be evaluated as alternatives or adjuncts to antibiotics as intramammary infusion or topical application to treat bovine mastitis, significantly improving the microbiological safety of milk.