We consider the impact of phosphatidic acid (namely, 1,2-dioleoyl-sn-glycero-3-phosphate, DOPA) on the properties of a zwitterionic (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) bilayer used as a model system for protein-free cell membranes. For this purpose, experimental measurements were performed using differential scanning calorimetry and the Langmuir monolayer technique at physiological pH. Moreover, atomistic-scale molecular dynamics (MD) simulations were performed to gain information on the mixed bilayer’s molecular organization. The results of the monolayer studies clearly showed that the DPPC/DOPA mixtures are nonideal and the interactions between lipid species change from attractive, at low contents of DOPA, to repulsive, at higher contents of that component. In accordance with these results, the MD simulations demonstrated that both monoanionic and dianionic forms of DOPA have an ordering and condensing effect on the mixed bilayer at low concentrations. For the DOPA monoanions, this is the result of both (i) strong electrostatic interactions between the negatively charged oxygen of DOPA and the positively charged choline groups of DPPC and (ii) conformational changes of the lipid acyl chains, leading to their tight packing according to the so-called “umbrella model”, in which large headgroups of DPPC shield the hydrophobic part of DOPA (the conical shape lipid) from contact with water. In the case of the DOPA dianions, cation-mediated clustering was observed. Our results provide a detailed molecular-level description of the lipid organization inside the mixed zwitterionic/PA membranes, which is fully supported by the experimental data.