Iron nanoparticles (FeNP) present excellent magnetic properties and chemical stability, and for this reason, they are often configured into materials for a variety of potential uses in medical, biotechnological, and other applications. In this work, iron oxide nanoparticles functionalized with galactomannan (FeNP/Gal) from Caesalpinia pulcherrima were synthesized and submitted to characterization and evaluation of the cytotoxic activity. The functionalized nanoparticles were synthesized by co-precipitation and subjected to a process of surface modification with galactomannan and epichlorohydrin. These nanomaterials were characterized using infrared spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential thermogravimetric analysis (DTA), and scanning electron microscopy (SEM). 1D and 2D nuclear magnetic resonance (NMR) spectroscopy were also used in the structural analysis of the galactomannan. In addition, in vitro study was carried out to evaluate the cytotoxic activity of the FeNP/Gal nanoparticles on human cells of the HEK-293 strain (ATCC® CRL-1573). The FeNP/Gal nanoparticles had an average diameter of 13 ± 2 nm as opposed to 11 ± 2 nm for unreacted FeNP. The infrared spectrum of the FeNP/Gal nanoparticles presents characteristic absorbance bands of their chemical constituents, confirming that the iron oxide nanoparticles were functionalized with galactomannan. The cytotoxicity assay for the FeNP/Gal nanoparticles did not show significant cytotoxicity against HEK 293-Human embryonic kidney cell lines below 800 µg/mL However, this study points out the possibility of using hemicellulose and other plant-based polysaccharides to produce nanostructured materials for tissue engineering and other biomedical applications.