This study has investigated the extraction process of hesperidin, a natural flavonoid found in the peel of two citrus fruits. In this research, for the first time, citrus peel wastes of bitter orange (Citrus aurantium) and Persian orange (Citrus reticulata) have been used to optimize the extraction of the bioflavonoid hesperidin. For this purpose, the effect of extraction temperature (45, 55, 65 °C), and ultrasonication time (0, 15, 30 min) (VC1-L2 Iran, 40kHz, 50W) in order to optimize the extraction of bioflavonoid hesperidin from citrus peel wastes of bitter orange and Persian orange with the hot methanol extraction method was investigated using the response surface method in the form of Box–Behnken design. The results of this study showed that the extraction temperature and ultrasound time were important and influential factors in hesperidin extraction efficiency. The optimal conditions for hesperidin extraction using bitter orange peel where the extraction temperature was 53.8 °C and the ultrasonication time was 21 min. Under these conditions, hesperidin extraction efficiency was reported as 4.08%. The results showed that the temperature and ultrasonication time had a significant effect on hesperidin extraction yield (p ˂ 0.05). Fourier transform infrared spectroscopy confirmed the presence of O–H, C=O, and C–H, C=C–C groups in the extracted sample (hesperidin). The results of the XRD test show that the creation of in-phase diffractions is due to the regular and crystalline structure of hesperidin. The results of the DSC test showed two endothermic peaks at temperatures of 103 and 251 °C. The results of the dynamic light scattering test showed that hesperidin crystals have an average particle size of 117.3 nm. The results of the antimicrobial test, which was performed on two bacteria, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922, by the agar well diffusion method, showed that S. aureus has more sensitivity than E.coli.