The present study investigates the role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes, NADPH oxidase and NO synthase, in the 1-methyl-3-octylimidazolium tetrafluoroborate ([omim][BF4])-mediated toxic mode of action in mussel hemocytes. Specifically, cell viability (using the neutral red uptake assay) was primarily tested in hemocytes treated with different concentrations of [omim][BF4] (0.1–10 mg L−1) and thereafter [omim][BF4]-mediated oxidative (in terms of superoxide anions/O2•- and nitric oxide/NO generation, as well as the enhancement of lipid peroxidation by-products, in terms of malondialdehyde/MDA) and genotoxic (in terms of DNA damage) effects were determined in hemocytes treated with 1 mg L−1 [omim][BF4]. Moreover, in order to investigate, even indirectly and non-entirely specific, the role of PI3-kinase, NADPH oxidase and NO synthase, the [omim][BF4]-mediated effects were also investigated in hemocytes pre-incubated with wortmannin (50 nM), diphenyleneiodonium chloride (DPI 10 μM) and NG-nitro-l-arginine methyl ester (l-NAME 10 μM), respectively. The results showed that [omim][BF4] ability to enhance O2•-, NO, MDA and DNA damage, via its interaction with cellular membranes, was significantly attenuated in the presence of each inhibitor in almost all cases. The current findings revealed for the first time that certain signaling molecules, such as PI3-kinase, as well as respiratory burst enzymes activation, such as NADPH oxidase and NO synthase, could merely attribute to the [omim][BF4]-mediated mode of action, thus enriching our knowledge for the molecular mechanisms of ILs toxicity.