Citrus oils (CO) have known antimicrobial properties and as such may serve as alternatives to conventional drug mastitis treatments; however, it is unknown how these oils affect environmental mastitis pathogens and the cow’s cellular immune response. The objectives of this study were to (1) determine the minimum inhibitory concentrations (MIC) of CO against Escherichia coli P4, and (2) evaluate CO cytotoxicity and their acute effect on oxidative response of bovine blood polymorphonuclear leukocytes (PMN). Citrus oils and its components, citral, linalool, valencene and limonlene, (0–10 µL/mL) were dissolved in ethanol and phosphate buffer solution with a 7:1 ethanol to oil ratio to maintain oil solubility. A control treatment of ethanol-only was also tested. Milk and blood were obtained from 12 healthy, mid-lactation dairy cows. Bacteriostatic MIC were determined through broth and milk microdilution. Bovine PMN were isolated from blood and incubated with varying citral, linalool, and ethanol concentrations. Following incubation, oil and ethanol cytotoxicity and PMN oxidative response were determined by quantifying PMN lactate dehydrogenase release into media and reactive oxidative species production, respectively. Cytotoxicity and oxidative burst response data were analyzed by ANOVA using the MIXED procedure of SAS 9.4. Of the CO components, citral and linalool had the lowest MIC and were unaffected by the presence of ethanol. Citral was the most effective at inhibiting (0.4 µL/mL in broth; 0.8 µL/mL in milk) E. coli P4 growth. No citral, linalool, or ethanol concentrations affected PMN oxidative burst response (P > 0.05); however, citral and linalool concentrations (0.1–0.8 µL/mL) were more toxic to PMN than control (0 µL/mL; P < 0.01). The ability of citral and linalool to inhibit proliferation of E. coli P4 highlight their potential as alternative antimicrobial therapies for bovine mastitis. Increased in vitro cytotoxicity suggests that further tests may be needed to optimize treatment strategy.