The increasing number of infections caused by pathogenic bacteria has severely affected human society. More and more deaths were originated from Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) infection each year. The potential and excellent bacteriostatic activity and resistance to biofilm formation of pillar[5]arene with different functional groups attract important attention to further study the relationship between antimicrobial activity and cytotoxicity by varying the length of the hydrophobic chain, the number of positive charges, and the hydrophobic/hydrophilic balance of the molecule. In this work, four pyridinium-based cationic pillar[5]arene (PPs) with linear aliphatic chains of different lengths were synthesized. After systematic characterization, their inhibition activities against S. aureus were investigated. It revealed that PP6 (six methylenes in each linker) exhibited excellent inhibition activity against S. aureus (ATCC 6538) with a minimum inhibitory concentration (MIC) of 3.91 μg/mL and a minimum bactericidal concentration (MBC) of 62.50 μg/mL. As expected, PP6 exhibited the strongest antibiofilm ability and negligible antimicrobial resistance even after the 20th passage. A study of the action mechanism of selected PPs on the bacterial membrane depolarization and permeability by transmission electron microscopy (TEM) disclosed that the cationic pyridine groups of PPs inserted into the negatively charged bacterial membranes, thereby leading to membranolysis, cytoplasmic content leakage, and cell death. Importantly, PPs all showed very low toxicity to mammalian cells (L929 and HBZY-1), which provided a significant reference for the construction of hypotoxic antibacterial biomaterials for multiple drug-resistant bacteria based on pyridinium-grafted cationic macrocycles with controllable hydrophobic chain lengths.