Background: Rhododendron adamsii (Sagaan Dalya) is a medical plant widely distributed in the Altai region, Mongolia, and Russia. It is traditionally used for its calming, restorative, and immune-boosting properties. Inflammation is a complex immune response against pathogens such as bacteria, viruses, and fungi, involving macrophages, fibroblasts, mast cells, and neutrophils. These cells release inflammatory mediators such as nitric oxide (NO), TNF-α, and IL-1β. In collaboration with the Russian Foundation for Basic Research, a project was initiated to investigate the bioactive fractions of Rh. rosea (L.) and Rh. adamsii and their effects on the production of TLR4 signaling end products and associated protein activation. Previous studies within this project have shown that certain bioactive fractions exhibit anti-inflammatory activity. Specifically, fraction 7.11 suppressed NO production and inflammatory signaling molecules in LPS-stimulated RAW 264.7 macrophages, while fractions 7.46 and 7.52 influenced the phosphorylation of proteins such as ERK1/2, JNK, and IRF3. These findings suggest the need for further investigation into the effects of Rh. adamsii bioactive fractions on inflammatory signaling pathways. Aim: This study aims to evaluate the effects of bioactive fractions derived from Rhododendron adamsii on the production of TLR4 signaling end products in RAW264.7 macrophage cell cultures. Materials and Methods: The study was conducted using RAW264.7 macrophage cell cultures and bioactive fractions extracted from Rh. adamsii, dividing the experiments into three groups. After stabilization, RAW264.7 cells were stimulated with 100 ng/mL LPS. Based on previous studies, non-toxic concentrations of bioactive fractions (10 µg/mL) were applied. NO production was measured using the Griess assay, while TNF-α and IFN-β cytokine levels were evaluated using ELISA. Each experiment were repeated three times, and data were statistically analyzed using SPSS 25.0, applying One-way ANOVA and Independent Samples T-test. Results: The NO production in the positive control group was significantly higher compared to the negative control. In contrast, the experimental groups showed a statistically significant reduction in NO production, suggesting a potential inhibitory effect on TLR4 signaling in macrophages. However, fraction 7.48 reduced TNF-α levels while increasing IFN-β production, but these changes were not statistically significant. Similarly, fraction 7.55 slightly reduced TNF-α and IFN-β levels, but the effect was also statistically unsignificant. Conclusion The bioactive fractions 7.48 and 7.55 of Rh. Adamsii reduced nitric oxide (NO) production in LPS-stimulated macrophage cell line cultures, suggesting that they may inhibit TLR4 signaling. However, their lack of effect on TNF-α and IFN-β production indicates that they do not influence TLR4 signaling mediated by these cytokines. Instead, they may affect the final product output through other pathways or different stages of signal transduction.