The aggregation behavior of mixed micellar solutions based on cationic hexadecylpiperidinium surfactants containing one or two hydroxy substituents and nonionic surfactant Tween 80 have been investigated by tensiometric, fluorimetric, and dynamic light scattering methods. The values of the critical micelle concentration were determined at varying component ratio. Based on the data obtained and their analysis using the Clint’s and Rubingh’s models one can conclude about a negative deviation from the ideal mixing (synergistic effect). It has been found out that the piperidinium surfactants, both in individually form and in mixed compositions with 0.5 molar fraction of nonionic surfactant showed high antimicrobial activity comparable to that of commercial antibiotics, while the presence of Tween 80 significantly reduced the acute toxicity of the system. A study of the membranotropic properties of piperidinium surfactants and the mixed systems, as well as an analysis of their effect on the permeability of the cell wall and cytoplasmic membrane of Staphylococcus aureus suggested that the antimicrobial effect was not associated with the destruction of the cell membrane, but was due to specific interactions of surfactants with cell components.