Reduction in atmospheric sulfur and intensified agriculture have led to sulfur deficiency, often correlated with a higher susceptibility to pathogens. The spread of fungal pathogens, such as the soil-born Verticillium longisporum, was observed. Defense responses of infected plants are linked to sulfur-containing compounds including glucosinolates (GSLs). Some pathogens infect their hosts at specific time periods during the day. To investigate the relation of sulfur-containing metabolites with diurnal effects of infection time points, Brassica napus plants cultivated at two different sulfur supplies, were infected with V. longisporum at four different time points during the day. It was demonstrated that 3, 7 and 14 days after inoculation the infected plants differed in their infection rate depending on the time point of infection. Additionally, infected plants had higher contents of sulfur-containing metabolites, such as specific GSLs, in comparison to non-infected plants. Sufficient sulfur fertilization was always reflected in higher contents of sulfur-containing compounds as well as a lower rate of infection compared to sulfur-deprived plants. On the microscopic level vascular occlusions in the hypocotyl were visible and the amount was dependent on the time point of infection. The results might be used to optimize sulfur fertilization to reduce susceptibility to V. longisporum.