This is the first report showing that the organic fraction of oil sands process water (OSPW-OF), and commercial naphthenic acids (C-NAs), cause immunotoxicity. The exposure of mouse bone marrow-derived macrophages (BMDM) to different amounts of C-NAs or OSPW-OF, did not affect cell viability in vitro. We examined whether exposure of BMDM to C-NAs or OSPW-OF affected various antimicrobial responses of these cells. A dose-dependent decrease in nitric oxide response was observed after treatment of BMDM with OSPW-OF, but not with C-NAs. Although OSPW-OF and C-NAs both down-regulated the respiratory burst response of BMDM, the suppression of the production of reactive oxygen intermediates was more pronounced in cells treated with OSPW-OF. Treatment with OSPW-OF or C-NAs reduced BMDM phagocytosis of zymosan and latex beads. The decrease of BMDM antimicrobial response after exposure to OSPW-OF or C-NAs, was accompanied by decreased pro-inflammatory cytokine gene expression. Oral exposure of mice to OSPW-OF caused down-regulation in the expression of genes encoding pro-inflammatory cytokines IFNγ, IL-1β and CSF-1. Our findings indicated that OSPW causes immunotoxic effects that may impair the ability of an exposed host to defend against infectious disease. Furthermore, given the differences between the effects of OSPW-OF and C-NAs, C-NAs should not be assumed to be a direct surrogate for the immunotoxic chemical species in OSPW.