Salicylic acid content and benzoic acid 2-hydroxylase (BA2H) activity were investigated in tobacco Bel-W3 and Bel-B leaves after exposure to tropospheric ozone in the conditions of ambient air. Plants were exposed in accordance with a standard methodology for ozone biomonitoring, in a three-year experiment. Free salicylic acid (SA), conjugated with glucose (SAG), and as a product of the BA2H activity was quantified with HPLC. In order to evaluate ozone injuries of leaves, an open source image analysis software was employed. Plants exposure to ambient ozone resulted in enhanced BA2H activity and intensified salicylic acid biosynthesis in leaves of Bel-W3 cultivar showing visible ozone injuries. The BA2H activity significantly correlated with SAG for ozone-exposed Bel-W3 plants. Both injuries and salicylic acid biosynthesis rate depended on the growth phase of leaves and nearly linear correlation between SA content and injuries was found for particular leaves of Bel-W3.