Green synthesis of nanoparticles (NPs) is eco-friendly, biocompatible, cost-effective, and highly stable. In the present study, Citrus sinensis peel extract was utilized to the fabrication of superparamagnetic iron oxide nanoparticles (SPIONs). The fabricated SPIONs were first characterized using UV–Visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Transmission electron microscopy (TEM), and vibrating sample magnetometer (VSM). The UV–Vis spectra analysis displayed a peak at 259 nm due to the surface plasmon resonance. The FTIR spectrum showed bands at 3306 cm⁻¹, and 1616 cm⁻¹ revealed the protein’s involvement in the development and capping of NPs. TEM analysis indicated that green synthesized SPIONs were spherical in shape with particle size of 20–24 nm. Magnetization measurements indicate that the synthesized SPIONs exhibited superparamagnetic behavior at room temperature. The antimicrobial activity, minimum inhibitory concentration (MIC), antioxidant potential, anti-inflammatory effect, and catalytic degradation of methylene blue by SPIONs were investigated in this study. Results demonstrated that SPIONs had variable antimicrobial effect against different pathogenic multi-drug resistant bacteria. At the highest concentration (400 μg/mL), SPIONs showed inhibition zones (14.7–37.3 mm) against all the target isolates. Furthermore, the MIC of synthesized SPIONs against Staphylococcus aureus, Streptococcus mutans, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, and Candida albicans were 3, 6.5, 6.5, 12.5, 50, 25 μg/mL, respectively. SPIONs exhibited strong antioxidant, anti-inflammatory, and catalytic dye degradation activities. Interestingly, Fe₃O₄ SPIONs shows optimum magnetic hyperthermia (MHT) techniques under an alternating magnetic field (AMF) measured in specific absorption rate (SAR) of 164, 230, and 286 W/g at concentrations 1, 5, and 10 mg/mL, respectively. Additionally, these newly fabricated SPIONs virtually achieve significant execution under the AMF in fluid MHT and are suitable for biomedical applications.