Invasive fungal infections have high morbidity and mortality rates and have become one of the most serious threats to human health. In the present study, a series of triazole antifungal derivatives with phenylthiophene backbone were obtained by structural modification of the lead compound using Iodiconazole as the lead compound. Among them, compound 19g is a triazole antifungal compound with 4-chloro-2-fluoro phenylthiophene backbone, which showed optimal antifungal activity against Candida albicans, Cryptococcus neoformans, and Aspergillus, with a MIC 80 value of 0.0625 μg/mL. In addition, compounds 19e, 19f, 19g, 19h, 19i and 19k exhibited different levels of inhibitory activity against fluconazole-resistant strains with MIC 80 values ranging from 0.0625 μg/mL to 32 μg/mL. Since compound 19g had optimal in vitro antifungal activity, we selected 19g for human liver microsomal stability and CYP enzyme inhibition assays as well as further evaluated the inhibitory activity of compound 19g on normal and cancerous cells in humans. Finally, we verified the inhibitory effect of compound 19g on the filamentation of Candida albicans and determined the mechanism of action by sterol composition analysis.