Tinospora cordifolia (Willd.) Miers is a common medicinal plant used in Ayurvedic medicine from which we have previously shown promising anti-inflammatory properties can be attributed to alkaloids in the plant. The objective of our study was to isolate and characterize anti-inflammatory alkaloids from this plant through guided bioassay fractionation and isolation of the methanolic stem extract. Methanolic extract was fractionated into hexane, chloroform, ethyl acetate, and aqueous extracts sequentially. We evaluated the extracts for inhibition of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The best extracts underwent chromatographic purifications and active compounds were identified using NMR and LC-MS. Two alkaloids decreased NO formation with an IC₅₀ of 18.3 µg/mL and 21.6 µg/mL with little if any cytotoxic activity. These data support the therapeutic application of selected T. cordifolia alkaloids as natural anti-inflammatory compounds and warrant pharmacological development.