Viscum album L., popularly known as mistletoe, is well known for its anti-cancer properties, and the pharmaceutical application of hydroalcoholic dry extracts is still limited due to its low solubility in aqueous media, and physicochemical instability. The Pluronic® F127 is an amphiphilic polymer, which permits the solubilization of lipophilic and hydrophilic compounds. In this investigation, physicochemical features of hydrogel containing V. album dry extract (VADE-loaded-hydrogel) were performed by: dynamic light scattering (DLS), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and transmission electron microscopy (TEM). VADE-loaded-hydrogel presented nanometer-size micelles with volume distribution ranging from 10.58 nm to 246.7 nm, and a polydispersity index of 0.441. The sample thermal analyses (TG and DSC) showed similar decomposition curves; however, the thermal events indicated an increase in thermal stability in relation to the presence of the extract. In addition to these interesting pharmaceutical features, IC50 values of 333.40 µg/mL and >1000 µg/mL were obtained when tumor (SCC-25) and non-tumor (L929) cells were incubated with VADE-loaded-hydrogel, respectively. The optical and ultrastructural cellular analysis confirmed the tumor selectivity since the following alterations were detected only in SCC-25 cells: disorganization of plasmatic membrane; an increase of cytoplasmatic vacuole size; alteration in the cristae mitochondrial shape; and generation of amorphous cellular material. These results emphasize the promising antitumoral potential of VADE-loaded-hydrogel as an herbal drug delivery system via in vitro assays.