Abstract: In order to discover compounds with higher antifungal activity and elucidate structure-activity relationship of this class of compounds, a series of butenolide oxime ether derivatives containing 5,5-gem dimethyl moiety were designed and synthesized using 5,5-dimethyl butenolide as the lead structure. Their structures were characterized by ¹H NMR, ¹³C NMR and HRMS. The in vitro antifungal activity evaluation revealed that the inhibition rates of compound D13 against Rhizoctonia solani, D8 against Sclerotinia sclerotiorum, D11 and D24 against Botrytis cinerea were 71.2%, 69.0%, 71.7% and 72.3% at the concentration of 50 mg/L, respectively. As for B. cinerea, the inhibition rates of compounds D5 - D30 with 4-pyridine moiety were much higher than those of compounds with substituted benzene at 4-position of butenolide, indicating the pyridine group can improve antifungal activities. These results provide insights into the structure-activity relationship and identify candidates with potent antifungal activity.