Bacteria are constantly developing greater antibiotic resistance; therefore, safe, efficacious, and cost-effective antibiotics are urgently needed. Ionic liquids (ILs) show great promise as antimicrobial agents because of their ability to disrupt bacterial membranes. In this study, a library of choline carboxylic acid-based ionic liquids were synthesized and utilized in vitro to discern their antimicrobial activity against Escherichia coli and methicillinresistant Staphylococcus aureus (MRSA). Here, choline was combined with carboxylic acids of varying alkyl chain lengths, degrees of saturation, and mole ratios and then added to E. coli and MRSA at different concentrations to determine the minimal bactericidal concentration (MBC). The MBC is linked to the presence of the anion because, when the cation is held constant, increasing the alkyl chain to its peak length, doubling the mole ratio, and removing the π bonds with respect to the anion tend to decrease MBC values, illustrating greater killing efficacy. This investigation provides insight into how the identity of the anion impacts the biocompatibility of the IL and the growth inhibition and morphology of the bacteria, ultimately establishing this class of materials as an effective, human-safe, and low-cost option for eradication of Gram-positive and Gram-negative bacteria.