Hydrogels of poly(vinyl alcohol) (PVA)/sodium alginate (SA), and magnetic nanoparticles (MNPs) were prepared by solvent casting in the absence and in the presence of magnets, in order to obtain MNPs distributed randomly (PVA/SA-rMNP) and magnetically oriented MNPs (PVA/SA-gMNP) in the polymer matrix. Atomic force microscopy (AFM) and magnetic force microscopy (MFM) techniques were used to evaluate the topography and to map the distribution of magnetic domains in the polymer matrix, respectively. The tip-surface distance (lift distance) of 50 nm during the MFM analyses facilitated the mapping of magnetic domains because the van der Waals forces were minimized. The magnetic signal stemming from clusters of MNPs were more easily identified than that from isolated MNPs. PVA and SA, PVA/SA, PVA/SA-rMNP, and PVA/SA-gMNP coatings with surface roughness (Ra) values of 3.8 nm, 28.7 nm, and 49.8 nm, respectively, were tested for the proliferation of mouse hippocampal HT-22 cells. While PVA/SA, PVA/SA-rMNP, and PVA/SA-gMNP coatings preserved cell viability >70% in comparison to the control (plastic plate) over 48 h, cell proliferation tended to decrease on surfaces with higher Ra values (PVA/SA-gMNP). These findings showed that the orientation of magnetic domains led to an increase of surface roughness, which decreased the viability of HT-22 cells. Thus, these results might be interesting for situations, where the control of cell proliferation is necessary.