Polyene macrolides are a large family of natural products typically produced by soil actinomycetes. Polyene macrolides are usually biosynthesized by modular and large type I polyketide synthases (PKSs), followed by several steps of sequential post-PKS modifications such as region-specific oxidations and glycosylations. Although known as powerful antibiotics containing potent antifungal activities (along with additional activities against parasites, enveloped viruses and prion diseases), their high toxicity toward mammalian cells and poor distribution in tissues have led to the continuous identification and structural modification of polyene macrolides to expand their general uses. Advances in in-depth investigations of the biosynthetic mechanism of polyene macrolides and the genetic manipulations of the polyene biosynthetic pathways provide great opportunities to generate new analogues. Recently, a novel class of polyene antibiotics was discovered (a disaccharide-containing NPP) that displays better pharmacological properties such as improved water-solubility and reduced hemolysis. In this review, we summarize the recent advances in the biosynthesis, pathway engineering, and regulation of polyene antibiotics in actinomycetes.