Superparamagnetic scaffolds with tailored magnetic response and hyperthermia efficiency are highly researched for biomedical applications, including nanomedicines and cancer therapy. However, the hyperthermia efficiency of such composites/scaffolds has always been investigated with respect to the amount of magnetic nanoparticles (MNPs), undermining the importance of MNP’s assembly and arrangement, particularly when the MNPs are immobilized within the matrices/scaffolds. This study achieves two drastically different arrangements of iron oxide-based MNPs in the bacterial nano-cellulose matrix, keeping the final MNP content the same: (i) Dispersed chains and (ii) Highly aggregated clusters, by varying the iron-ion concentrations with simultaneous oleic acid (OA) capping. Consequently, two significantly different hyperthermia performances were obtained at the same fraction of MNPs: (i) a rapid rise to 76 ℃ in MNP chains and (ii) a gradual rise to 40 ℃ in MNP clusters in the 900 s. This is attributed to substantial enhancement in the effective magnetic anisotropy constant owing to the in-situ governed chain assembly of MNPs in the nanofibrous reactor. The highly stable performance (>180 days), along with room-temperature superparamagnetism and excellent biocompatibility of both the m-BC assemblies, pave the way for tunable hyperthermia and drug delivery applications. Nevertheless, assembly-driven tailored hyperthermia observed in this study underscores the need to evaluate the magnetic scaffolds (or MNPs) under practical biological conditions for accurate quantifications.