Hot pepper (Capsicum annuum) represents one of the most widespread functional foods of the Mediterranean diet, and is associated with a reduced risk of developing cardiovascular disease, cancer, and mental disorders. In particular, its bioactive spicy molecules, named Capsaicinoids, exhibit polypharmacological properties. Among them, Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the most studied and reported in variegated scientific contributions for its beneficial effects, often linked to mechanisms of action unrelated to the activation of Transient Receptor Potential Vanilloid 1 (TRPV1). In this study, we present the application of in silico methods to Capsaicin for evaluating its inhibitory activity against the tumor-associated human (h) expressed CA IX and XII. In vitro assays confirmed Capsaicin inhibitory activity towards the most relevant tumor-relatedhCA isoforms. In particular, thehCAs IX and XII showed an experimental KIvalue of 0.28 μM and 0.064 μM, respectively. Then, an A549 model of non-small cell lung cancer, typically characterized by an elevated expression ofhCA IX and XII, was employed to test the inhibitory effects of Capsaicin in vitro under both normoxic and hypoxic conditions. Finally, the migration assay revealed that Capsaicin [10 µM] inhibits cells from moving in the A549 cells model.