In this study, three types of hydrophobized alkyl-modified magnetic nanoparticles (MNPs) comprising direct alkylated-MNPs (A-MNPs), silica-mediated alkyl MNPs (A-SiMNPs), and arginine (Arg)-mediated alkyl MNPs (A-RMNPs) were synthesized successfully. For this purpose, the co-precipitation method was used to synthesize, and octadecyl trimethoxy silane (OTMS) was used as a functionalizing agent. Accordingly, the hydrophobic octadecyl moieties were connected to MNPs. The nanoparticles (NPs) were characterized by XRD, SEM, FTIR, CHN, DLS, and zeta potential analyses. The synthesized coated MNPs represented a decrease in surface charge and magnetization alongside increased surface hydrophobicity and size. It was revealed that the alkylation process was successfully performed to all three MNPs, but A-SiMNPs showed the highest hydrophobicity. Additionally, the novel A-RMNPs, as the most biocompatible type, and A-MNPs showed the highest magnetization among the synthesized MNPs. The results indicate that synthesized NPs can play an important role in bio applications. However, it was revealed that alkyl chains are easily connected to all three MNPs, and that A-MNPs contained the highest alkyl chains and could affect the re-folding and denaturation process of recombinant proteins.