Tomato gray leaf spot is caused by three necrotrophic species of the genus Stemphylium. Secondary metabolites (SMs) are a highly diverse set of low molecular mass bioactive compounds, which have been associated to different biological roles. We hypothesized that S. lycopersici isolates that differ on disease severity and their ability to sporulate synthetize a pool of soluble SMs that are different. Each isolate was grown on V8 and PDA plates at 25°C for 14 days. These cultures were lyophilized and used as source of soluble metabolites, being an aqueous extract obtained through sonication and ultrafiltration (0.22 μm pore-size membrane). Spectroscopic analysis of each extract was performed (UV?Vis absorption spectra and fluorescence?excitation-emission matrices). Fitotoxicity of extracts was evaluated in vitro by means of a detached leaflet assay. While the most of extracts showed an absorbance maximum at 450 nm, any absorbance peak was found for the extract from CIDEFI-213 isolate grown on V8 medium. However, fluorescence intensity and emission regions were different according both the fungi and medium used. A higher fluorescence was found in CIDEFI-216 extracts.