Atractylis flava Desf. (AF) is common plant that is widely used for its anti- inflammatory and antioxidant properties. The purpose of this study was, therefore, to evaluate the cytotoxic effect and the molecular basis of antioxidant and anti-inflammatory effects of the ethyl acetate extract (AFEAE) obtained from the whole plant A. flava. This was accomplished through the use of NR8383 alveolar rat macrophage cells. Cultures of alveolar rat macrophage cells were treated with AFEAE (25–800 μg/mL), and cell viability was determined via WST-1 and LDH tests. In turn, the gene expression levels of nuclear factor κB (NF-κB), tumor necrosis factor-alpha (TNFα), interleukin 1 beta (IL1-β), interleukin 6 (IL-6), mitochondrial dynamin like GTPase (OPA1), Succinate dehydrogenase complex subunit A (SDHA) and neutrophil cytosolic factor 1 (NCF1) were assessed by applying RT-qPCR. The results show that ethyl acetate extracts of A. flava have non-cytotoxic effects, and the gene expression analysis demonstrates that AFEAF extracts generate significant downregulation of NF-κB, TNFα, IL-1 β, IL-6, NCF1, OPA1 and SDHA, compared to untreated cells. This study reveals that Atractylis flava ethyl acetate extract administration may be considered as a potential therapeutic strategy for inflammatory related diseases.