Epidemics of basil downy mildew (DM) incited by Peronospora belbahrii have been very severe in Italy since 2013, in part due to the very favorable weather conditions, and losses have occurred in many commercial farms, even after repeated mefenoxam treatments. DM populations from basil plants and seeds, which are associated with failure in downy mildew control under field and greenhouse conditions, have been tested for their sensitivity to mefenoxam. Basil plants were inoculated with a sporangial suspension of seven DM populations and treated, before and after inoculation with the pathogen, with different dosages of mefenoxam: 100 µg/ml, which corresponds to the currently applied field dosage, 200 µg/ml and 1000 µg/ml. Azoxystrobin was used at the field dosage as the chemical control. Three out of four DM populations from seeds and two out of three from basil plants were found to be able to infect basil plants in the presence of 100 µg/ml and 200 µg/ml of mefenoxam, while the field dosage of azoxystrobin (186 µg/ml) was found to be completely effective. The sensitive populations of P. belbahrii were completely controlled by the field dosage of both chemicals also 14 days after the last treatment. This study provides new information on the potential risk of introducing mefenoxam-resistant P. belbahrii inoculum in the field through seeds infected by resistant strains.