Objectives: Our study reports the immunomodulatory potency of Quercus infectoria gall extract in vitro. The aqueous extract was prepared and examined for its effects on cell proliferation, phagocytic activity, nitric oxide (NO) production, and cytokine synthesis by murine macrophages. Methods: Proliferative, phagocytic activity, and NO production of extract-treated and control cells were studied using proliferative assay, flow cytometry, and Griess reaction, respectively. An enzyme-linked immunosorbent assay was performed to determine the levels of pro- and anti-inflammatory cytokines in the macrophage culture. Results: Treated macrophages had a higher proliferative rate and phagocytic activity compared to untreated macrophages. The cell treatment with an extract concentration of 64 μg/mL demonstrated a significant decrease in NO production (p < 0.001). An increase in cytokine levels (IL-2, IL-5, IL-10, IL-17A, IL-23, TGF-β1) was observed; however, this increase was not statistically significant. Conclusions: Our study suggests that gall extract possesses the potential for augmenting immunomodulatory activity by cellular mediated mechanism and could play a role in regulating the innate immune response.