Three fluorinated cationic surfactants were prepared by condensing N-methyl diethanol amine pentafluoro benzoate with stoichiometric amounts of octyl, dodecyl or hexadecyl bromide. The surface properties and parameters were investigated to find the relationship between the structures of the hydrophobic portion of such compounds. The properties studied include surface tension, critical micelle concentration (CMC), effectiveness (Πcmc), maximum surface excess (Γmax) and minimum surface area (Amin) were investigated with respect to different concentrations at 25 °C. Standard free energies of micellization and adsorption of the prepared surfactants in the aqueous solution were studied. The values of Γmax, standard free energies of micellization $$ \Updelta {\text{G}}_{\text{mic}}^{\text{o}} $$ and adsorption $$ \Updelta {\text{G}}_{\text{ads}}^{\text{o}} $$ were found to increase with the chain length, while the cmc and minimum surface area occupied by one molecule Amin were found to decrease. The biocidal activity was determined through the inhibition zone diameter of prepared compounds which were measured against five strains of a representative group of microorganisms.