Muscle-invasive bladder cancer (MIBC) is a common form of BC in dogs. Adjuvant chemotherapy administration is commonly applied in MIBC cases, but patients sometimes experience treatment failure and recurrence. Therefore, supplements with anticancer properties, such as traditional Chinese medicines (TCMs), are required, and they have been widely used in Japanese human medicine and may be useful in veterinary medicine. Furthermore, organoid cultures can mimic the characteristics of their original tissues, such as self-renewal and organization. We previously established a novel experimental model for MIBC using a dog BC organoid (DBCO) culture. Herein, we examined the antiproliferative effects and mechanisms of 39 substances, consisting of TCMs, TCM supplements, and crude drug extracts, on DBCOs. Among the TCMs, D3 (also known as Shibe-ria), which is a mixture of chaga (Inonotus obliquus) and notoginseng (Panax notoginseng), significantly diminished the cell viability of DBCOs. The expression of BC stem cell markers, CD44 and SOX2, was reduced considerably in the D3-treated DBCOs. Among the components of D3, chaga exerted an antiproliferative effect on DBCO, whereas notoginseng did not. The administration of D3 also significantly reduced the volume of DBCO xenografted tumors in mice in vivo. Overall, D3 may have benefits as a natural anticancer supplement in veterinary medicine.