The intriguing characteristics of nanoparticles have fueled recent advancement in the field of nanotechnology. In the current study, a microbial-based bioflocculant made from the SCOBY of Kombucha tea broth was purified, profiled, and utilized to biosynthesize iron nanoparticles as a capping and reducing agent. UV–visible absorption spectroscopy, transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDX), and TGA were used to characterize the Fe nanoparticles. The FT-IR spectra showed functional groups such as hydroxyl, a halogen (C-Br), and carbonyl, and the alkane (C-H) functional groups were present in both samples (bioflocculant and FeNPs) with the exception of the Fe-O bond, which represented the successful biosynthesis of FeNPs. The TEM investigation revealed that the sizes of the produced iron nanoparticles were between 2.6 and 6.2 nm. The UV-vis spectra revealed peaks at 230 nm for the bioflocculant and for the as-fabricated FeNPs, peaks were around 210, 265, and 330 nm, which confirms the formation of FeNPs. X-ray diffraction presented planes (012), (104), (110), (113), (024), (116), and (533) and these planes correspond to 17.17, 32.58, 33.75, 38.18, 45.31, 57.40, and 72.4° at 2Ө. The presence of Fe nanoparticles presented with 0.82 wt% from the EDX spectrum of the biosynthesized FeNPs. However, Fe content was not present from the bioflocculant. SEM images reported cumulus-like particles of the bioflocculant, while that of FeNPs were agglomerated and hexagonal with sizes between 18 and 50 nm. The TGA of FeNPs showed thermal stability by retaining above 60% of its weight at high temperatures. It can therefore be deduced that the purified bioflocculant produced by a yeast Pichia kudraivzevii can be utilized to synthesize FeNPs with the current simple and effective method.