Cercospora leaf spot (CLS) is a serious sugar beet disease caused by the fungus Cercospora beticola that affects plants all over the world and causes significant yield losses in all cultivation areas. The main purpose of this study was to evaluate the antifungal activity of n-alkyl dimethyl benzyl ammonium chloride (n-ADBAC) and Bacillus subtilis against C. beticola, a pathogen causing Cercospora leaf spot of sugar beet, both in vitro and in vivo. Antifungal activity of n-ADBAC was tested in laboratory experiments against the pathogen’s growth at different concentrations, including 0.25, 0.50, 1.00, 1.50 and 2.00 g/L of water. At all concentrations examined, n-ADBAC was able to stop or limit the pathogen’s mycelial development. While the concentration of 2.00 g/L had a strong effect on pathogen growth inhibition (68.15%), it also had antifungal activity. Moreover, the results of antagonist assay of B. subtilis showed that it inhibits the growth of the causal pathogen, with an inhibition rate of up to 84.62%. Data also showed that application of n-ADBAC and B. subtilis as foliar treatments decreased the disease severity of Cercospora leaf spot under greenhouse and field conditions compared to untreated plants. The results revealed that n-ADBAC was the only treatment that consistently improved sugar beet agronomic characteristics, such as root yield and sugar content in roots compared to control plants. Data also showed that the tested treatments reduced the percentage of non-sugar substances, i.e. sodium, potassium and alpha-amino nitrogen in the roots.