Yeasts are among the best-studied experimental organisms. They are widely used as model systems in various research fields, including genetics, molecular biology, biochemistry, and cell biology. Beyond basic research, yeasts have significant applications in biotechnology and industry for the production of value-added bioproducts, including yeast extracts, enzymes, lipids, pigments, antioxidants, and others. Additionally, yeasts are some of the most utilized “cell factories” for the production of recombinant proteins with various applications. Yeasts were among the model systems used to evaluate the effects of pulsed electric fields (PEF) on living cells, specifically examining the main characteristics of the temporary or permanent loss of membrane barrier properties under the influence of an applied electric field, a process termed electroporation or electropermeabilization. Along with the general characteristics of this process established with different types of organisms, yeasts exhibit several morphological and functional differences, which determine specific features of electroporation, their ability to release intracellular contents, and the possibility of electroloading various extracellular components. Currently, electroporation (electropermeabilization) of yeast is predominantly used to enable the transport of specific substances across the cell membrane for both basic research and various biotechnological applications. Some of the earliest experiments on loading molecules into intact yeast, as well as the most common current application of this technique, focus on the introduction and expression of plasmid DNA. This process, known as electrotransformation, involves electrical treatment that induces reversible membrane permeabilization, ensuring that the yeast remain viable and capable of reproduction. On the other hand, irreversible electropermeabilization, which leads to the release of intracellular content, represents a very promising alternative approach for the production of value-added low-molecular substances, as well as for the selective recovery of native and recombinant proteins with different applications.