The microbial P450s perform an array of oxidative and other chemical reactions that are both crucial for the viability of the bacterial, archaeal, and fungal hosts, and which have numerous important applications. The soluble nature of the bacterial and archaeal P450s has facilitated their expression and purification in high yields, and has enabled the determination of the crystal structures of several important members of the P450 superfamily. Many of the major breakthroughs in our knowledge of the catalytic mechanisms of the P450s have been made through spectroscopic and transient kinetic studies on the microbial P450s, including recent research that has definitively identified the highly reactive P450iron–oxo species compound I and has demonstrated its catalytic potency. This chapter describes our current knowledge on the structural and functional properties of the microbial P450s, including their involvement in pathways for production of industrially important molecules such as antibiotics. Aspects such as engineering of these P450s for novel reaction chemistry are also detailed, along with emerging data for novel P450 enzymes fused to both redox and nonredox partner proteins, and novel systems that bypass the requirement for redox partner proteins altogether. The microbial P450s provide a rich source of catalysts that continue to provide new information on the versatility of the P450s as well as novel activitieswith applications in the biomedical, biofuels, and bioremediation fields. This chapter highlights recent breakthroughs and developments that will ensure that microbial P450s remain center stage for biotechnology applications in the coming years.