Pyrrolidinium-type small molecule ionic liquids (ILs), poly(ionic liquid) (PIL) homopolymers, and their corresponding PIL membranes were synthesized and used for antibacterial applications. The influences of substitutions at the N position of pyrrolidinium cation on the antimicrobial activities against both Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were studied by minimum inhibitory concentration (MIC). The antibacterial efficiency of both the small molecule ILs and PIL homopolymers increased with the increase of the alkyl chain length of substitutions. Furthermore, PIL homopolymers show relatively lower MIC values, indicating better antimicrobial activities than those of the corresponding small molecule ILs. However, the antibacterial properties of the PIL membranes are contrary to corresponding ILs and PIL homopolymers, which reduce with the increase of alkyl chain length. Furthermore, the resultant PIL membranes show excellent hemocompatibility and low cytotoxicity toward human cells, demonstrating clinical feasibility in topical applications.