Tilia americana var. mexicana is used in Mexican traditional medicine to treat anxiety and inflammatory processes. Several glycosides derived from quercetin and kaempferol, including tiliroside, isoquercetin, and quercetin-3-β-D-glucoside, were reported as the main anxiolytic compounds in this species; to our knowledge, compounds with anti-inflammatory effects have not been previously described. In this study, whole plants were obtained from rooted cuttings with indole-3-butyric acid (IBA) under greenhouse conditions. Multiple shoots and callus cultures were established from apical and axillary buds from T. americana var. mexicana cuttings. The apical buds (75%) were the best explant for shoot induction (2–3 shoots per explant) on Murashige and Skoog (MS) medium supplemented with 2.0 mg L−1 of 6-benzyl aminopurine plus 0.25 mg L−1 α-naphthaleneacetic acid. Callogenesis occurred in both types of buds in the treatments constituted by thidiazuron with 0.1 mg L−1 IBA. High-performance liquid chromatography analysis of leaves and callus methanolic extracts allowed the identification of quercetin-3-β-D-glucoside and tiliroside anxiolytic compounds, and of the scopoletin anti-inflammatory compound. The methanolic leaf and callus extracts showed anti-inflammatory activities in a 12-O-tetradecanoylphorbol-13-acetate-induced ear edema model with median effective doses (ED50) of 0.38 and 1.73 mg per ear for the leaf and callus extracts, respectively.