A compound containing an imidazole moiety and a 2,4-dienone motif with significant activity toward several fungi was discovered in a screen for new antifungal compounds. Then, a total of 26 derivatives of this compound were designed, synthesized and evaluated through in vitro and in vivo antifungal activity assays. Several compounds exhibited improved antifungal activities compared to the lead compound. Of the derivatives, compounds 31 and 42 exhibited strong, broad-spectrum inhibitory effects toward Candida spp. In particular, the two derivatives exhibited potent antifungal activities toward the fluconazole-resistant isolate C. albicans 64110, with both having MIC values of 8 µg/mL. In addition, they had significant inhibitory effects toward two Gram-positive bacteria, Staphylococcus aureus UA1758 (compound 31: MIC = 8 µg/mL; compound 42: MIC = 4 µg/mL) and Staphylococcus epidermidis UF843 (compound 31: MIC = 8 µg/mL; compound 42: MIC = 8 µg/mL). The results of an animal experiment indicated that both compounds could improve the survival rate of model mice infected with ATCC 90028 (fluconazole-susceptible isolate). More importantly, the two compounds exhibited notable in vivo effects toward the fluconazole-resistant C. albicans isolate, which is promising with regard to the clinical problem posed by fluconazole-resistant Candida species.