Antimicrobial preservative compounds are added to foods to target specific pathogens and spoilage organisms. White mustard essential oil (WMEO) is an extract that contains 4-hydroxybenzyl isothiocyanate, a compound which has been demonstrated to have antimicrobial activity in limited studies. The objective of this research was to determine the in vitro antimicrobial activity of WMEO against gram-positive and gram-negative spoilage and pathogenic bacteria and determine the effect of food components on the antimicrobial activity. The bacteria Escherichia coli, Salmonella enterica serovar Enteritidis, Enterobacter aerogenes, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Lactobacillus fermentum, as well as the acid- and preservative-resistant yeast Schizosaccharomyces pombe, were evaluated. All microorganisms were inhibited by WMEO at 8.3 g/liter (equivalent to 1,000 mg/liter 4-hydroxybenzyl isothiocyanate). In general, WMEO was more effective against gram-negative than against gram-positive bacteria. Salmonella Enteritidis and S. pombe were the most sensitive, with inhibition at as low as 2.1 g/liter. The effects on growth profiles varied but included increased lag phases and lethality, indicating both bacteriostatic and bactericidal activity. Soybean oil had a negative effect on the efficacy of WMEO against L. monocytogenes, and at 5% soybean oil, the antimicrobial activity against Salmonella Enteritidis was eliminated after 48 h. Sodium caseinate at 1% also negated the antimicrobial effect of WMEO against Salmonella Enteritidis and decreased its effectiveness against L. monocytogenes. The presence of starch had no significant effect on the antimicrobial activity of WMEO against L. monocytogenes and Salmonella Enteritidis. Thus, WMEO is effective against a wide range of microorganisms and has potential to be used in foods, depending upon the target microorganism and food components present.