In order to develop a safer and more effective preservative for pickled mustard tubers, it is necessary to examine the antimicrobial activities, applications and underlying mechanisms of potassium sorbate (PS) and N-α-lauroyl-L-arginine ethyl ester monohydrochloride (LAE) as well as their mixture. In this research, spoilage bacteria were isolated from the rotten mustard tuber and used as the research object. In vitro antimicrobial experiments based on the checkerboard assay were performed to determine the minimum inhibitory concentration (MIC) of LAE and PS at different pHs, and the joint antimicrobial effect was examined and verified in the preservation of pickled mustard tuber. In addition, taking the most dominant isolated spoilage microorganisms as the research object, the peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) activities, the DPPH free radical scavenging rate, the protein content, and the malondialdehyde (MDA) content were determined under the action of LAE. The major spoilage microorganisms isolated were used as the test bacteria (inducing Virgibacillus halodenitrificans, Pseudomonas stutzeri and Penicillium janthinellum). The results showed that the MICs of LAE and PS were 16~32 mg/L and 256~1024 mg/L, respectively, with PS exhibiting shows a positive correlation between antimicrobial activities and acidity, and LAE acting oppositely. PS and LAE exhibited an additive or synergistic effect in the antimicrobial activities towards the test bacteria. The combined use of LAE and PS could inhibit (by 1 to 2 orders of magnitude) the proliferation of the microorganisms in packed pickled mustard tubers. Under the press of LAE, the POD, CAT and SOD activities, DPPH radical scavenge rate, protein content and MDA content decreased. LAE and PS can effectively inhibit the spoilage microorganisms in pickled mustard tuber, and their mix has a promising potential of enhancing preservation performance.Furthermore, the potential antimicrobial mechanisms of LAE might involve the decrease of membrane integrity and increase of oxidative stress of the microbial cells.