Abstract This study was performed to validate the previous antimicrobial and cytotoxic data on the influence of ionic liquids as coatings of silver nanoparticles (AgNPs). The antibacterial and cytotoxicity assessments were carried out against different microorganisms and a cancerous cell line. AgNPs with two different ionic-liquid coatings and hydrocarbon chains were synthesized and characterized. We tested the antibacterial activity of these NPs against Salmonella typhi, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Candida albicans in planktonic forms and against Enterococcus faecalis and Escherichia coli in biofilm forms. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was employed for toxicity evaluation. The antimicrobial activity of NPs with 12 carbons was significantly higher than those with 18 carbons. Furthermore, NPs with 12 carbons were also effective against bacterial biofilms. All of the NPs tested had good cell viability at different antimicrobial concentrations. The length of the hydrocarbon chain is an essential factor in determining the antimicrobial activity of ionic-liquid-coated AgNPs. The variation in ionic-liquid coatings was not as effective as other influencing factors. Evaluation of AgNPs using other alkyl chain lengths to find the optimal size is recommended.