This study was conducted to evaluate the attenuating ability of diclazuril treatment on Eimeria species mix oocyst during and after sporulation for protection of layer chicks against Eimeria infection. Field isolates of Eimeria oocysts were collected and propagated in chickens to obtain a continuous source of oocysts. The collected un sporulated oocysts divided into two parts: the first part treated by diclazuril 20% during sporulation for 48h, while, the second part subjected for sporulation firstly, then treated by diclazuril 20% for 48h. The treated oocysts used in immunization of layer chicks at 4^th day of age. Chicks were divided into 6 groups: two groups inoculated by 20% diclazuril treated oocysts. The third group immunized by commercial live vaccine (Coccivac D) and fourth group inoculated by un treated oocyst. Two control groups (control infected un immunized group and control un infected un immunized group). At 21 day of age, chicks were challenged by  7.5 x10⁴ oocyst. The post immunization evaluation showed significant decrease in bloody diarrhea score, lesion score and oocyst shedding of diclazuril treated and commercial vaccine groups compared to the group inoculated by un treated oocyst. While the post challenge evaluation revealed a significant decrease in oocyst per gram (OPG) count, lesion value and bloody diarrhea score with improved weight gain in diclazuril treated groups compared to commercial vaccine group and control infected un immunized group. Also, diclazuril treatment showed excellent anticoccidial indices compared to commercial vaccine and un treated oocysts groups. The results of this study proved that diclazuril 20% concentration is effective in attenuation of Eimeria species oocyst and induced a significant protactive effect against challenge in layer chicks.