A series of new spiro-substituted pyranocoumarin derivatives have been synthesized starting from the commercially available 7-hydroxycoumarin and the conformation of the pyran ring was investigated. The antioxidant activity of the compounds was evaluated in-vitro, by means of three different tests: the interaction with the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), the competition with DMSO for hydroxyl radicals scavenging ability and the quenching of superoxide anions generated by the enzymic xanthine–xanthine oxidase system. In the DPPH test the spiroadamantane derivative 13 was the most active and possessed a 40% inhibition at a concentration of 400 μm. All compounds successfully compete with DMSO for hydroxyl radicals generated by the Fe3+/ascorbic acid system. Compound 13 inhibited the oxidation of DMSO (3.125mm) by 93% at 2 mm and by 71% at 0.25 mm. The corresponding second-order rate constants have been estimated and all compounds demonstrated higher rate constants compared with the reference compounds, 7-hydroxycoumarin and mannitol. Derivatives possessing extended conjugation showed the highest inhibitory activity for superoxide anions generated by the xanthine–xanthine oxidase system, although the results of this experiment possessed partial parallelism with the results observed in the other two tests. The overall obtained data indicate that the size of the different spiro- substituents influence the degree of free radical scavenging and demonstrate the importance of extended conjugation for the anti-oxidant activity. Due to its multiple mechanism of protective action, derivative 13 may serve as a lead for the development of analogues that could be useful for the treatment of pathophysiological processes dependent upon reactive oxygen species.