Biogenic selenium nanoparticles (SeNPs) have been shown to exhibit increased bioavailability. Fermentation of pollen by a symbiotic culture of bacteria and yeasts (SCOBY/Kombucha) leads to the release of pollen content and enhances the prebiotic and probiotic effects of Kombucha. The aim of this study was to fortify Kombucha beverage with SeNPs formed in situ by Kombucha fermentation with pollen. Response Surface Methodology (RSM) was used to optimize the biosynthesis of SeNPs and the pollen-fermented Kombucha beverage. SeNPs were characterized by Transmission electron microscopy energy-dispersive X-ray spectroscopy (TEM-EDX), Fourier-transform infrared spectroscopy (FTIR), Dynamic light scattering (DLS), and Zeta potential. The pollen-fermented Kombucha beverage enriched with SeNPs was characterized by measuring the total phenolic content, antioxidant activity, soluble silicon, saccharides, lactic acid, and the total content of Se0. The polyphenols were identified by liquid chromatography–mass spectrometry (LC-MS). The pollen and the bacterial (nano)cellulose were characterized by scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), FTIR, and X-Ray diffraction (XRD). We also assessed the in vitro biocompatibility in terms of gingival fibroblast viability and proliferation, as well as the antioxidant activity of SeNPs and the pollen-fermented Kombucha beverage enriched with SeNPs. The results highlight their increased biological performance in this regard.