Animal venoms are a complex mixture of highly specialized toxic molecules. Among them, pore-forming proteins (PFPs) or toxins (PFTs) are one of the major disease-causing toxic elements. The ability of the PFPs in defense and toxicity through pore formation on the host cell surface makes them unique among the toxin proteins. These features made them attractive for academic and research purposes for years in the areas of microbiology as well as structural biology. All the PFPs share a common mechanism of action for the attack of host cells and pore formation in which the selected pore-forming motifs of the host cell membrane-bound protein molecules drive to the lipid bilayer of the cell membrane and eventually produces water-filled pores. But surprisingly their sequence similarity is very poor. Their existence can be seen both in a soluble state and also in transmembrane complexes in the cell membrane. PFPs are prevalent toxic factors that are predominately produced by all kingdoms of life such as virulence bacteria, nematodes, fungi, protozoan parasites, frogs, plants, and also from higher organisms. Nowadays, multiple approaches to applications of PFPs have been conducted by researchers both in basic as well as applied biological research. Although PFPs are very devastating for human health nowadays researchers have been successful in making these toxic proteins into therapeutics through the preparation of immunotoxins. We have discussed the structural, and functional mechanism of action, evolutionary significance through dendrogram, domain organization, and practical applications for various approaches. This review aims to emphasize the PFTs to summarize toxic proteins together for basic knowledge as well as to highlight the current challenges, and literature gap along with the perspective of promising biotechnological applications for their future research.