Pseudo-oligosaccharides are microbial-derived secondary metabolites whose chemical structures contain pseudosugars (glycomimetics). Owing to their high resemblance to the molecules of life (carbohydrates), most pseudo-oligosaccharides show significant biological activities. Some of them have been used as drugs to treat human and plant diseases. Because of their significant economic value, efforts have been put into understanding their biosynthesis, optimizing their fermentation conditions, and engineering their metabolic pathways to obtain better production yields. Many unusual enzymes participating in diverse biosynthetic pathways to pseudo-oligosaccharides have been reported. Various methods and conditions to improve the production yields of the target compounds and eliminate byproducts have also been developed. This review article describes recent studies on the biosynthesis, fermentation optimization, and metabolic engineering of high-value pseudo-oligosaccharides.