Epiphytic lichen biodiversity (LB) distribution was evaluated in a 1000 km2 area in Tuscany (Central Italy). In the same area, a survey was performed to monitor tropospheric ozone (O3) phytotoxic effects using tobacco plants (Nicotiana tabacum) cv. Bel-W3 (O3 supersensitive) and cv. Bel-B (O3 resistant) as bioindicators. The LB proved to be negatively correlated with ambient levels of nitrogen oxides, particulate and carbon monoxide, but not with benzene. LB spatial distribution followed a peculiar trend, with highest values in areas with low population density. Data from an O3 analyser set in evidence a typical circadian profile, confirming the photochemical nature of this pollutant. Quite high nocturnal values along the coast were related to the presence of surrounding mountains which constrained the back-and-forth flow of the air in sea breezes. Long- and short-term phytotoxicity critical levels for O3 were systematically trespassed. Bel-W3 tobacco was always affected in every site involved in the study; average O3 injury distribution was greater in rural areas; tobacco response was positively correlated to several O3 descriptors with second-order functions. Correlation analysis failed to demonstrate any association between lichen and tobacco data, due to the fact that LB values were higher in inland zones, far from the main urban and industrial areas, where instead damage to tobacco plants was generally lower. It is concluded that the lichen biodiversity is not suitable for monitoring O3 levels, at least in the study area.