Cytochrome P450 monooxygenases are versatile catalysts that selectively oxidize C-H bonds. By mutating an active site threonine to glutamate, P450 monooxygenases can be transformed into peroxygenases that can use H2O2 for catalysis. This eliminates the need for expensive co-factors or electron transfer partner proteins. This threonine residue is highly conserved and is important in the mechanism of oxygen activation by these enzymes. The cytochrome P450 enzyme CYP102A1 (P450BM3) is a highly adaptable monooxygenase tailored through protein engineering for diverse chemical synthesis applications. Mutating the threonine 268 residue to glutamate (Thr268Glu) transformed the enzyme’s heme domain into a peroxygenase that uses H2O2. This variant displayed higher peroxide-driven hydroxylation activity towards fatty acids ranging from undecenoic to hexadecenoic acid compared to the wild-type heme domain, with oxidation occurring mainly at the ω-1 through ω-3 positions during fatty acid oxidation. The Thr268Glu peroxygenase enzyme hydroxylated 10-undecenoic aid at the allylic ω-2 carbon in a selective manner. Isotopic substitution with [9,9,10,10-d4]-dodecanoic acid showed a kinetic isotope effect (kH/kD) ranging from 7.9 to 9.5, slightly lower in the peroxygenase system. This suggests that for both the monooxygenase and peroxygenase systems, C-H bond abstraction is important, indicating that compound I, the ferryl-oxo radical cation intermediate, is the probable reactive intermediate in both systems. The per oxygenase variant simplifies cytochrome P450 systems for oxidations, demonstrated by the oxidation of tetradecanoic acid using light-driven H2O2 generation. The Thr268Glu mutant was also used to investigate the products arising from the P450-catalyzed oxidation of selected aromatic substrates. We also produced and tested the further mutated heme domain variant (R47L/ A82F/ F87V/ L188Q/ T268E or BM3muTE), which contains additional mutations which are known to enhance the oxidation of aromatic substrates in the P450Bm3 holoprotein. The Thr268Glu mutant showed higher catalytic activity for the oxidative hydroxylation of tetralone, oxidising this substrate regioselectively. The product formation activity for aromatic substrates was much higher for Thr268Glu than the BM3muTE by a wide margin. Next, we generated a new cytochrome P450 peroxygenase catalyst using the bacterial CYP154C8 enzyme from a Streptomyces bacterium. Mutating the threonine on the I-helix to glutamate transforms this monooxygenase enzyme into a peroxygenase. This enabled the hydroxylation of unactivated carbon-hydrogen bonds in complex steroid molecules in a selective manner. Selective biocatalytic formation of the 16α-hydroxy steroid metabolite was possible using this engineered bacterial cytochrome P450 enzyme. CYP154C8 enzyme could regio- and stereo-selectively hydroxylate steroids such as progesterone and androstenedione at the C16 position. The CYP116B subfamily of cytochrome P450s, known for self-sufficiency, shows promise in biotechnology applications. These enzymes are reported to catalyse a range of challenging oxidative reactions without additional protein partners. Their heme domain has also been demonstrated to have a high tolerance level for hydrogen peroxide. Here, we assessed cytochrome P450 monooxygenase CYP116B46 from the thermophilic Tepidiphilus thermophilus. Investigation of the substrate range for this enzyme revealed that its optimal substrate was 2-hydroxyphenylacetic acid, and this was selectively oxidised to homogentisic acid (2,5-dihydroxyphenyl acetic acid). We also evaluated the potential of the T278E mutation of the CYP116B46 heme domain to transform this enzyme into a more proficient peroxygenase. This mutant produced the highest amount of product using 2-hydroxyphenylacetic acid as a substrate at 50 °C, confirming the enhanced peroxygenase activity resulting from the T278E mutation in CYP116B46 and the thermophilic nature of the enzyme.