Quaternary chitosan derivative with covalently bonded antioxidant (QCG) was used as media for synthesis of selenium nanoparticles (SeNPs). SeNPs were characterized using AFM, TEM, and DLS methods. The data confirmed the formation of stable nanoparticles with a positive charge (34.86–46.73 mV) and a size in the range 119.5–238.6 nm. The antibacterial and fungicidal activity of SeNPs occurred within the range of values for chitosan derivatives. In all cases, the highest activity was againstC. albicans(MIC 125 µg/mL). The toxicity of the modified selenium nanoparticles to eukaryotic cells was significantly higher. Among nanoparticle samples, SeNPs that were synthesized at 55 °C demonstrated the highest toxicity against Colo357 and HaCaT cell lines. Based on these results, SeNPs loaded with doxorubicin were obtained. DOX loading efficiency was about 18%. QCG-SeNPs loaded with DOX at a concentration of 1.25 μg/mL inhibited more than 50% of hepatocarcinoma (Colo 357) cells and about 70% of keratinocytes (HaCaT).