Aberrant activation of the Hedgehog (HH) signaling is a critical driver in tumorigenesis. The Smoothened (SMO) receptor is one of the major upstream transducers of the HH pathway and a target for the development of anticancer agents. The SMO inhibitor Vismodegib (GDC-0449/Erivedge) has been approved for treatment of basal cell carcinoma. However, the emergence of resistance during Vismodegib treatment and the occurrence of numerous side effects limit its use. Our group has recently discovered and developed novel and potent SMO inhibitors based on acylguanidine or acylthiourea scaffolds. Here, we show that the two acylguanidine analogs, compound (1) and its novel fluoride derivative (2), strongly reduce growth and self-renewal of melanoma cells, inhibiting the level of the HH signaling target GLI1 in a dose-dependent manner. Both compounds induce apoptosis and DNA damage through the ATR/CHK1 axis. Mechanistically, they prevent G2 to M cell cycle transition, and induce signs of mitotic aberrations ultimately leading to mitotic catastrophe. In a melanoma xenograft mouse model, systemic treatment with 1 produced a remarkable inhibition of tumor growth without body weight loss in mice. Our data highlight a novel route for cell death induction by SMO inhibitors and support their use in therapeutic approaches for melanoma and, possibly, other types of cancer with active HH signaling.