Two chromone C-glucosides, biflorin (1) and isobiflorin (2), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of 1 and 2 on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages were evaluated, and 1 (IC50 = 51.7 and 37.1 μM, respectively) was more potent than 2 (IC50 > 60 and 46.0 μM). The suppression of NO and PGE2 production by 1 correlated with inhibition of iNOS and COX-2 protein expression. Compound 1 reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound 1 inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, 1 reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, 1 reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound 1 showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of 1.