The interactions of bacteria with their environment are governed by a complex interplay between biological and physicochemical phenomena. The main challenge is the joint determination of the intertwined interfacial characteristics of bacteria such as mechanical and hydrodynamic softness, interfacial heterogeneity, and electrostatic properties. In this study, we have combined electrokinetics and force spectroscopy to unravel this intricate coupling for two types of Shewanella bacterial strains that vary according to the nature of their outer, permeable, charged gel-like layers. The theoretical interpretation of the bacterial electrokinetic response allows for the estimation of the hydrodynamic permeability, degree of interfacial heterogeneity, and volume charge density for the soft layer that constitutes the outer permeable part of the bacteria. Additionally, the electrostatic interaction forces between an AFM probe and the bacteria were calculated on the basis of their interfacial properties obtained from advanced soft particle electrokinetic analysis. For both bacterial strains, excellent agreement between experimental and theoretical force curves is obtained, which highlights the necessity to account for the interfacial heterogeneity of the bioparticle to interpret AFM and electrokinetic data consistently. From the force profiles, we also derived the relevant mechanical parameters in relation to the turgor pressure within the cell and the nature of the bacterial outer surface layer. These results corroborate the heterogeneous representation of the bacterial interface and show that the decrease in the turgor pressure of the cell with increasing ionic strength is more pronounced for bacteria with it thin surface gel-like layer.