This research represents a deliberate choice of larger ligands to regulate agglomeration, resulting in the creation of Fe3O4 nanoparticles with the precise shape control. The reaction environment was controlled by examining the often-overlooked factors, such as the use of surfactants, heat pre-treatment, and degassing. Through the use of cetyltrimethylammonium bromide (CTAB), along with the appropriate heat treatment and degassing, cubic-shaped particles (average length of 55 ± 5 nm) with a single crystalline structure were successfully generated. Substituting CTAB with other surfactants like dodecylammonium iodide (DDAI) and dodecylammonium chloride (DDAC) led to the production of irregular shapes. However, when a combination of CTAB and DDAC (2:1 M ratio) or CTAB and DDAI (4:1 M ratio) was employed, spherical particles with an average diameter of 70 ± 10 nm and rod-shaped particles with an average length of 100 ± 20 nm were obtained, respectively. All IO-B, IO-BC, and IO-BI particles exhibited superparamagnetic properties.