Malonyl-coenzyme A (CoA) is a crucial extender unit for the synthesis of mycolic and other fatty acids in mycobacteria, generated in a reaction catalyzed by acetyl-CoA carboxylase. We previously reported on the essentiality of accD6Mtb encoding the functional acetyl-CoA carboxylase subunit in Mycobacterium tuberculosis. Strikingly, the homologous gene in the fast-growing, non-pathogenic Mycobacterium smegmatis - (accD6Msm) appeared to be dispensable, and its deletion did not influence the cell lipid content. Herein, we demonstrate that, despite the difference in essentiality, accD6Msm and accD6Mtb encode proteins of convergent catalytic activity in vivo. To identify an alternative, AccD6-independent, malonyl-CoA synthesis pathway in M. smegmatis, a complex genetic approach combined with lipid analysis was applied to screen all five remaining carboxyltransferase genes (accD1-accD5) with respect to their involvement in mycolic acid biosynthesis and ability to utilize acetyl-CoA as the substrate for carboxylation. This approach revealed that AccD1Msm, AccD2Msm and AccD3Msm are not essential for mycolic acid biosynthesis. Furthermore, we confirmed in vivo the function of AccD4Msm as an essential, long-chain acyl-CoA carboxyltransferase, unable to carboxylate short-chain substrate. Finally, our comparative studies unambiguously demonstrated between-species difference in in vivo ability of AccD5 carboxyltransferase to utilize acetyl-CoA that influences AccD6 essentiality in pathogenic and non-pathogenic mycobacteria.