The Mycobacterium tuberculosis enhanced intracellular survival (Eis_Mtb) protein is a clinically important aminoglycoside (AG) multi-acetylating enzyme. Eis homologues are found in a variety of mycobacterial and non-mycobacterial species. Variation of the residues lining the AG-binding pocket and positions of the loops bearing these residues in the Eis homologues dictates the substrate specificity and, thus, Eis homologues are Nature-made tools for elucidating principles of AG recognition by Eis. Here, we demonstrate that the Eis from Anabaena variabilis (Eis_Ava), the first non-mycobacterial Eis homologue reported, is a multi-acetylating AG-acetyltransferase. Eis_Ava, Eis from Mycobacterium tuberculosis (Eis_Mtb), and Eis from Mycobacterium smegmatis (Eis_Msm) have different structures of their AG-binding pockets. We perform comparative analysis of these differences and investigate how they dictate the substrate and cosubstrate recognition and acetylation of AGs by Eis.