Magnetite nanoparticles (Fe3O4NPs) are widely tested in various biomedical applications, including magnetically induced hyperthermia. In this study, the influence of the modifiers, i.e., urotropine, polyethylene glycol, and NH4HCO3,on the size, morphology, magnetically induced hyperthermia effect, and biocompatibility were tested for Fe3O4NPs synthesized by polyol method. The nanoparticles were characterized by a spherical shape and similar size of around 10 nm. At the same time, their surface is functionalized by triethylene glycol or polyethylene glycol, depending on the modifiers. The Fe3O4NPs synthesized in the presence of urotropine had the highest colloidal stability related to the high positive value of zeta potential (26.03 ± 0.55 mV) but were characterized by the lowest specific absorption rate (SAR) and intrinsic loss power (ILP). The highest potential in the hyperthermia applications have NPs synthesized using NH4HCO3, for which SAR and ILP were equal to 69.6 ± 5.2 W/g and 0.613 ± 0.051 nHm2/kg, respectively. Their application possibility was confirmed for a wide range of magnetic fields and by cytotoxicity tests. The absence of differences in toxicity to dermal fibroblasts between all studied NPs was confirmed. Additionally, no significant changes in the ultrastructure of fibroblast cells were observed apart from the gradual increase in the number of autophagous structures.