This work was conducted to investigate the effects of the polyethylene oxide (PEO) as a template on the structures, functional groups, magnetic properties, and antibacterial activities of Fe₃O₄/PEO/TMAH ferrofluids. Synthesis of Fe₃O₄ nanoparticles was carried out by using the co-precipitation method obtained from iron sand. The ferrofluid synthesis was done by coating Fe₃O₄ nanoparticles with tetramethylammonium hydroxide (TMAH) and dispersed in H₂O. Based on the structural analysis, it was known that all samples had a magnetite phase with particle sizes ranging from 7.8 to 10.5 nm. The Fe₃O₄/PEO/TMAH ferrofluid functional groups presented appropriate bonds of Fe₃O₄ as a filler, TMAH as a surfactant, and H₂O as a liquid carrier. The magnetic characteristics of Fe₃O₄ nanoparticles and Fe₃O₄/PEO/TMAH ferrofluids indicated the superparamagnetic state. The saturation magnetization value of Fe₃O₄ nanoparticles and Fe₃O₄/PEO/TMAH ferrofluids decreased as increasing the molecular weight of polyethylene oxide ranging from 1000 to 20,000. Furthermore, the antibacterial activities performed by the dilution method identified that increasing the molecular weight of polyethylene oxide increased the antibacterial performance of the Fe₃O₄/PEO/TMAH ferrofluids against Escherichia coli and Bacillus subtilis.