The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for organism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tuberculosis. The sequence of CYP125A7 from M. marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed differences in the resting heme spin state and in the binding modes and affinities of certain azole inhibitors. CYP125A7 did not show a significant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some differences in the extent of the Type I shifts to the high-spin form with steroids and a higher affinity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identified as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria.Copyright © 2020 Elsevier Inc. All rights reserved.