Procambarus clarkii, an American crayfish, was introduced in the SW Iberian Peninsula three decades ago. This region has no native crayfishes; therefore, P. clarkii constitutes a potential threat for the embryos and larvae of amphibians, which may lack evolved defenses against it. We assessed the effects of this crayfish on the Natterjack Toad, Bufo calamita, using two complementary approaches. First, we studied the effect of crayfish presence on B. calamita breeding habitat use in temporary ponds of Doñana Natural Park (southwestern Spain). We surveyed 31 temporary ponds for the presence of P. clarkii and B. calamita eggs and tadpoles, and we conducted a logistic regression to determine if crayfish were significantly associated with Natterjack Toad distribution after habitat variables were taken into account. Second, we experimentally evaluated the impact of P. clarkii on B. calamita embryos in natural ponds with a two × two factorial experiment, using two ponds (one with and one without crayfish) and two treatments (embryos exposed or not exposed to predators). Crayfish presence was a negative predictor of B. calamita breeding habitat use; co-occurrence of the two species was limited to only one pond. Moreover, the predation experiment showed that survival of B. calamita embryos was strikingly reduced when they were directly exposed to crayfish compared to survival in all other treatments. Our results indicated that P. clarkii has a strong effect on B. calamita reproductive success and that it may play an important role in structuring amphibian communities in temporary ponds.