Continuous use of anticoccidial treatments against Eimeria infections has resulted in the development of drug resistance. This study aimed to evaluate the anticoccidial efficacy of a methanolic extract derived from the endemic Canary rue (Ruta pinnata) plant of the Canary Islands, Spain, against Eimeria ninakohlyakimovae using in vitro assays. Freshly unsporulated oocysts were exposed to different concentrations of R. pinnata extract and thereafter evaluated for sporulation inhibition. Additionally, anticoccidial activity was examined by testing the viability of the E. ninakohlyakimovae sporozoites and their ability to infect bovine colonic epithelial cells after incubation with different concentrations of R. pinnata plant extract. The inhibition of oocyst sporulation by the extract was both time and concentration dependent, with certain combinations affording the same levels of sporulation inhibition as formaldehyde used as positive control (P < 0.001). Moreover, concentrations >0.1 mg/mL also affected not only the viability of the sporozoites but also their cell invasion capacity (P < 0.001). Altogether, these results show that methanolic fruit extracts from R. pinnata have important anticoccidial activity against oocysts and sporozoites of Eimeria. The potential efficacy of the extracts against other animal/human parasites remains to be elucidated, and further studies are needed to better understand its mode of action against coccidian parasites.