Cold atmospheric plasma (CAP) is a tool with the ability to generate reactive oxygen and nitrogen species (RONS), which can induce therapeutic effects like disinfection, wound healing, and cancer treatment. In the plasma oncology field, CAP-treated hydrogels (PTHs) are being explored for the local administration of CAP-derived RONS as a novel anticancer approach. PTHs have shown anticancer effects in vitro, however, they have not yet been studied in more relevant cancer models. In this context, the present study explores for the first time the therapeutic potential of PTHs using an advanced in ovo cancer model. PTHs composed of alginate (Alg), gelatin (Gel), Alg/Gel combination, or Alg/hyaluronic acid (HA) combination are investigated. All embryos survived the PTHs treatment, suggesting that the in ovo model could become a time- and cost-effective tool for developing hydrogel-based anticancer approaches. Results revealed a notable reduction in CD44+ cell population and their proliferative state for the CAP-treated Alg-HA condition. Moreover, the CAP-treated Alg-HA formulation alters the extracellular matrix composition, which may help combat drug-resistance. In conclusion, the present study validates the utility of in ovo cancer model for PTHs exploration and highlights the promising potential of Alg-based PTHs containing HA and CAP-derived RONS for cancer treatment.