The continued depletion of oil reserves, a necessity for energy security and the environmental importance of reducing greenhouse gas emissions has prompted the industrial development of clean, non-conventional, renewable fuels. Transport fuels are currently composed primarily of fossil-derived alkanes and commonly contain a minor biofuel component of bioethanol in gasoline or fatty acid methyl esters (FAMEs) in diesel. Increasing biofuel supply to satisfy governmental targets imposes substantial technological constraints on automobile engine manufacturers and raises controversies over arable land usage. ‘Drop-in’, microbial, alkane biofuels are structurally identical to fossil fuels and their production does not compete directly with arable farming, thereby obviating the problems associated with the manufacture and deployment of bioethanol and FAMEs. This study investigated alkane biosynthesis in two non-photosynthetic bacteria, Mycobacterium sp. NCIMB 10403 and Desulfovibrio desulfuricans, to evaluate their potential for use as biocatalysts in the industrial manufacture of ‘drop-in’ biofuels. This study employed single- and two-dimensional gas chromatography-mass spectrometry as a means to provide a rigorous analysis of alkane biosynthesis in these bacteria. In addition, a microarray analysis was used to develop an understanding of the genes potentially important in regulating alkane production in D. desulfuricans. In contrast to the original reports from the 1960’s, Mycobacterium sp. NCIMB 10403 and D. desulfuricans NCIMB 8307 did not synthesise alkanes. Alkane biosynthesis was confirmed in D. desulfuricans NCIMB 8326 although the alkane quantities and carbon chain length distribution differed significantly to those previously reported. The microarray data gave evidence to suggest that the expression of genes encoding a long chain fatty acid-CoA ligase and an aspartyl/glutamyl-tRNA amidotransferase may be important for regulating alkane biosynthesis in D. desulfuricans. Furthermore, several genes encoding hypothetical proteins were identified as being potentially involved directly in the formation of alkanes.