Amphotericin B (AmB) is a widely used antifungal agent especially for the therapy of systemic fungal infections. However, the severe side effects of AmB often leads to the premature termination of the treatment. So it is imperative to find the drugs that can both reduce the dosage and enhance the antifungal efficacy of AmB. Here we demonstrated that Nicotinamide (NAM), a cheap and safe vitamin, could enhance the antifungal activities of AmB. We demonstrated the synergistic interaction of NAM and AmB against Candida albicans as well as other Candida spp. and Cryptococcus neoformans. Moreover, NAM could enhance of the activity of AmB against biofilm. This enhancement was also observed in disseminated candidiasis in vivo. Our further study revealed that AmB could induce oxidative damage through the modification of histone acetylation. AmB could inhibit the expression of HST3, an H3K56 deacetylase in C. albicans. The immunoblotting test revealed excessive H3K56ac in AmB-treated fungal cells. Consistantly, the hst3Δ mutant displayed high sensitivity to AmB, while addition of NAM, an H3K56 deacetylation inhibitor, resulted in an even severe inhibition in the growth of this strain. These results indicated that AmB could execute antifungal activity via boosting H3K56ac which was mediated by HST3, and the mechanism for the synergistic interaction of NAM and AmB was based on exacerbating this process, which led to even excessive H3K56ac and oxidative damage. This finding provided theoretical basis for better understanding the antifungal mechanisms of AmB and clinical application of this drug.