We previously demonstrated that aminoglycoside acetyltransferases (AACs) display expanded cosubstrate promiscuity. The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis is responsible for the resistance of this pathogen to kanamycin A in a large fraction of clinical isolates. Recently, we discovered that Eis is a unique AAC capable of acetylating multiple amine groups on a large pool of aminoglycoside (AG) antibiotics, an unprecedented property among AAC enzymes. Here, we report a detailed study of the acyl-coenzyme A (CoA) cosubstrate profile of Eis. We show that, in contrast to other AACs, Eis efficiently uses only 3 out of 15 tested acyl-CoA derivatives to modify a variety of AGs. We establish that for almost all acyl-CoAs, the number of sites acylated by Eis is smaller than the number of sites acetylated. We demonstrate that the order of n-propionylation of the AG neamine by Eis is the same as the order of its acetylation. We also show that the 6' position is the first to be n-propionylated on amikacin and netilmicin. By sequential acylation reactions, we show that AGs can be acetylated after the maximum possible n-propionylation of their scaffolds by Eis. The information reported herein will advance our understanding of the multiacetylation mechanism of inactivation of AGs by Eis, which is responsible for M. tuberculosis resistance to some AGs.