Vitamin D₃ is known to be a key component in the defense against Mycobacterium tuberculosis (Mtb) infection through the regulation of cytokine and effector molecules. Conversely, alcohol exposure has been recognized as an immune dysregulator. Macrophages were extracted from D₃ deficient and sufficient diet mice and supplemented with D₃ or exposed to ethanol during ex vivo infection using M. bovis BCG, as a surrogate for Mtb. Results of our study indicate that while exogenous supplementation or alcohol exposure did alter immune response, in vivo diet was the greatest determinant of cytokine and effector molecule production. Alcohol exposure was found to profoundly dysregulate primary murine macrophages, with ethanol-exposed cells generally characterized as hyper- or hyporesponsive. Exogenous D₃ supplementation had a normative effect for diet deficient host, however supplementation was not sufficient to compensate for the effects of diet deficiency. Vitamin D₃ sufficient diet resulted in reduced cell cytotoxicity for the majority of time points. Results provide insight into the ramifications of both the individual and combined health risks of D₃ deficiency or alcohol exposure. Given the clinical relevance of D₃ deficiency and alcohol use comorbidities, outcomes of this study have implications in therapeutic approaches for the treatment of tuberculosis disease.