Magnetic nanoparticles (MNPs) are prone to exhibit physicochemical changes caused by their interaction with biological solutions. However, such interactions have been less considered in cancer therapy studies. The behavior of four iron oxide MNP formulations with different surface coatings, namely, chitosan (CS), polyvinyl alcohol (PVA), carboxymethyldextran (CMX), and polydimethylamine (PEA), was investigated, after their exposure to four different cell culture media (DMEM/F12 and MEM, among others) and six different cancer cell lines (HT29, HT1080, T24, MDA-MB-231, BxPC-3, and LS174T). The sedimentation (V) and diffusion (V) velocities of MNPs in different culture media were calculated. Atomic absorption spectroscopy (AAS) and dynamic light scattering (DLS) were used to quantify cell uptake efficiency and physicochemical properties, respectively. Apart from PVA-coated MNPs, CMX-, CS-, and PEA-coated MNPs clustered and increased notably in size when dispensed in culture media. The different MNP formulations led either to a low (PVA-coated MNPs), medium (CS- and CMX-coated MNPs), or high (PEA-coated MNPs) clustering in the different culture media. Clustering correlated with the V and V of the MNPs and their subsequent interaction with cells. In particular, the CMX-coated MNPs with higher V and lower V internalized more readily than the PVA-coated MNPs into the different cell lines. Hence, our results highlight key considerations to include when validating nanoparticles for future biomedical applications.