Bipolaris sorokiniana is a dangerous pathogen of plants from Poaceae family. A secondary metabolite with carcinogenic properties produced by this fungus is sterigmatocystin. A field experiment with inoculation of the grain of 9 spring barley genotypes with B. sorokiniana No. 36 was carried out in the years 2012–2014 in the Zamość region. Field observations revealed leaf spot caused by B. sorokiniana in all studied genotypes. In 2012, values of the leaf infection index varied from 21.88 (Oberek) to 48.12 (Hajduczek), in 2013 from 25.31 (Skald, Oberek) to 50.00 (STH 7910) and in 2014 from 21.88 (Oberek) to 50.00 (Hajduczek). In the experimental combination with B. sorokiniana, colonies of this fungus in the years 2012–2014 accounted for: 81.22%, 93.11% and 71.78%, respectively, and in control combination: 40.06%, 32.26% and 33.72%, respectively. The chemical analysis of grain of 9 barley genotypes obtained from plants in an experimental combination with B. sorokiniana in 2014, revealed the presence of sterigmatocystin in the genotypes: Hajduczek, Kormoran, Stratus and STH 7910. The sterigmatocystin concentration ranged from 5.39 ng·g–1 (STH 7910) to 67.05 ng·g–1 (Hajduczek). A statistically significant correlation was found between the value of the leaf infection index and the concentration of sterigmatocystin in the grain.