Orange peel, which is a rich source of polyphenolic compounds, including hesperidin, is produced as waste in production. Therefore, optimization of the extraction of hesperidin was performed to obtain its highest content. The influence of process parameters such as the kind of extraction mixture, its temperature and the number of repetitions of the cycles on hesperidin content, the total content of phenolic compounds and antioxidant (DPPH scavenging assay) as well as anti-inflammation activities (inhibition of hyaluronidase activity) was checked. Methanol and temperature were key parameters determining the efficiency of extraction in terms of the possibility of extracting compounds with the highest biological activity. The optimal parameters of the orange peel extraction process were 70% of methanol in the extraction mixture, a temperature of 70 °C and 4 cycles per 20 min. The second part of the work focuses on developing electrospinning technology to synthesize nanofibers of polyvinylpyrrolidone (PVP) and hydroxypropyl-β-cyclodextrin (HPβCD) loaded with hesperidin-rich orange peel extract. This is a response to the circumvention of restrictions in the use of hesperidin due to its poor bioavailability resulting from low solubility and permeability. Dissolution studies showed improved hesperidin solubility (over eight-fold), while the PAMPA-GIT assay confirmed significantly better transmucosal penetration (over nine-fold). A DPPH scavenging assay of antioxidant activity as well as inhibition of hyaluronidase to express anti-inflammation activity was established for hesperidin in prepared electrospun nanofibers, especially those based on HPβCD and PVP. Thus, hesperidin-rich orange peel nanofibers may have potential buccal applications to induce improved systemic effects with pro-health biological activity.