Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used medications to treat conditions such as arthritis, pain, and fever. They reduce inflammation by inhibiting cyclooxygenase (COX) enzymes that catalyze the committed step in prostaglandin (PG) biosynthesis. Despite their significant therapeutic benefits, many NSAIDS have undesirable adverse effects. The aim of this study was to discover novel COX inhibitors from natural sources. Here, we describe the synthesis and anti-inflammatory activity of the COX-2 inhibitor axinelline A (<b>A1</b>), which was isolated from <i>Streptomyces axinellae</i> SCSIO02208, and its analogues. Compared to the synthetic analogues, the natural product <b>A1</b> has stronger COX inhibitory activity. Although <b>A1</b> is more active against COX-2 than COX-1, its selectivity index is low; therefore, it may be classified as a nonselective COX inhibitor. Its overall activity is comparable to the clinically used drug diclofenac. <i>In silico</i> studies showed that <b>A1</b> binds to COX-2 in a similar manner to diclofenac. Inhibition of COX enzymes by <b>A1</b> in LPS-stimulated murine RAW264.7 macrophages resulted in suppression of the NF-κB signaling pathway, leading to reduced expression of pro-inflammatory factors such as iNOS, COX-2, TNF-α, IL-6, and IL-1β and reduced production of PGE<sub>2</sub>, NO, and ROS. The potent <i>in vitro</i> anti-inflammatory activity of <b>A1</b>, together with its lack of cytotoxicity, makes it an attractive candidate for a new anti-inflammatory lead.