A series of 16 imidazolium-based surface-active ionic liquids (SAILs) were synthesized. The designed SAILs, consisted of either one amphiphilic center, 3-alkoxymethyl-1-benzylimidazolium or 3-alkoxymethyl-1-benzyl-2-methylimidazolium, and two amphiphilic centers, 3,3′-[α,ω-(dioxaalkane)]bis(1-benzylimidazolium) or 3,3′-[α,ω-(dioxaalkane)]bis(1-benzyl-2-methylimidazolium). These compounds were investigated in terms of thermal analysis, ability to self-organize to micelles, and antimicrobial activity. The insertion of an ether moiety in the alkyl side or spacer chain favored adsorption at the air–water interface and micellization in the bulk solution when compared to commercial chloride surfactants. The results showed that attachment of a methyl group to the imidazolium ring led to lower the critical micelle concentration (CMC) than lengthening the alkyl chain or spacer in the imidazolium cation. Additionally, biological activity increased with increasing hydrophobicity of the compounds. The methylation of the imidazole ring at positions 2 and 2′ yielded in a notable increase in bacteriostatic activity, especially against Gram-positive bacteria, notably the Staphylococcus aureus strains. It has been demonstrated that minor structural modifications, specifically substituting the imidazole ring instead of elongating the alkyl chain or spacer, can modify the micellization and aggregation behavior, leading to differences in both the efficacy and variety of antimicrobial activity of amphiphilic SAILs.