Atom-resolved noncontact atomic force microscopy (NC-AFM) was recently used to reveal that the insulating spinel MgAl2O4(100) surface, when prepared under vacuum conditions, adopts a structurally well-defined Al and O-rich structure (Al4-O4-Al4 termination) consisting of alternating Al and double-O rows, which are, however, interrupted by defects identified as interchanged Mg in the surface layers (so-called antisite defects). From an interplay of futher NC-AFM experiments and first-principles NC-AFM image simulations, we present here a detailed analysis of the NC-AFM contrast on the MgAl2O4(100) surface. Experiments show that the contrast on MgAl2O4(100) in atom-resolved NC-AFM is dominated by two distinctly different types of contrast modes, reflecting two oppositely charged tip-apex terminations. In this paper, we analyze the contrast associated with these imaging modes and show that a positively charged tip-apex (presumably Mg2+) interacts most strongly with the oxygen atoms, thus imaging the oxygen lattice, whereas a negatively charged tip-apex (O2−) will reveal the cation sublattice on MgAl2O4. The analysis of force-vs-distance calculations for the two tips shows that this qualitative picture, developed in our previous study, holds for all realistic tip-surface imaging parameters, but the detailed resolution on the O double rows and Al rows changes as a function of tip-surface distance, which is also observed experimentally. We also provide an analysis of the tip dependency and tip-surface distance dependency for the NC-AFM contrast associated with single Al vacancies and Mg-Al antisite defects on the MgAl2O4(100) surface and show that it is possible on the basis of NC-AFM image simulations to discriminate between the Al3+ and Mg2+ species in antisite defects and hypothetical Al vacancies.