In this study, we have fabricated the PHBV functionalized graphite oxide using freeze drying technique, followed by ‘in situ’ pay loading of Fe3O4 nanoparticles onto the hydrophobic plate of the composite basal plane; thereby, mechanically and thermally stable, bio-imaging Fe3O4/GO-g-PHBV composites have been developed. The synthesis of Fe3O4/GO-g-PHBV composite was confirmed by field emission SEM and TEM analyses, X-ray diffraction and Fourier transform infrared spectroscopy. The wrapping of PHBV copolymer into the graphene layers was investigated by atomic force microscopy and Raman spectral analyses which provided the shifting of the 2D band with low signal intensity in the range of 2600–3000 cm−1. The bactericidal activities of the Fe3O4/GO-g-PHBV composite films were found to exhibit more efficiency against Gram-negative bacteria strains compared to Gram-positive strains. In vibrating sample magnetometer (VSM) analysis, the zero value of coercivity revealed the super-paramagnetic nature of the Fe3O4/GO-g-PHBV composites. The Phantom agar magnetic resonance imaging analysis revealed the efficiency of Fe3O4 nanoparticles as a negative contrast (T2 contrast) along with higher relaxivity value. The significant fibroblast cell (NIH 3T3) adhesion and proliferation (85%) on the Fe3O4/GO-g-PHBV composite surface indicated the physiological and biocompatible stability of that composite along with the presence of large π conjugated aromatic domain. © 2018 Elsevier B.V.