Four series of spiro[benzoxazine-piperidin]-one derivatives were designed and synthesized. Their inhibition percentages against chitin synthase and antifungal activities were evaluated. Based on the preliminary biological assays, the series of derivatives containing α, β-unsaturated carbonyl fragment which had moderate to excellent CHS inhibitory activity and antifungal activity were further researched. In this series of compounds, eight out of twenty-one compounds had good to excellent inhibitory activity against chitin synthase with an inhibition percentage value above 60% at the concentration of 300 μg/mL. Among them, compounds 9a, 9o, 9s and 9t showed excellent chitin synthase inhibitory activity with IC50 values of 0.14 mM, 0.11 mM, 0.10 mM and 0.16 mM, respectively, which were equal to that of the control drug (polyoxin B). The results of sorbitol protection assays and evaluation of antifungal activity against micafungin-resistant fungi further proved that the target of these synthesized compounds was chitin synthase. The antifungal activity evaluation showed that compounds 9a, 9d, 9h, 9s and 9t had broad-spectrum antifungal activity in vitro and their antifungal activities are equal to those of fluconazole and polyoxin B. The result of combination use showed this series of compounds combined with fluconazole had additive or synergistic effects. In addition, compounds 9a, 9o and 9t showed good antifungal activity against fluconazole-resistant C. albicans and fluconazole-resistant C. neoformans variants. Consequently, the results showed that these compounds were chitin synthase inhibitors and antifungal agents and had significant activity against drug-resistant fungal variants.