People are known to utilize plants for treatment of many diseases. Cycadales are one of these ancient plants that contained different active constituents. Due to the lack of adequate research on Cycadales, we conducted this study. The current study investigated and compared different biological activities for four different plants. Cycas thouarsii, Cycas pectinata, Dioon spinulosum and Encephalartos laurentianus were tested for the first time for cytotoxic, antioxidant and antimicrobial activities. Dioon spinulosum displayed significant cytotoxic activity on VERO cell line that was higher than 5-Fluorouracil. D. spinulosum possessed the highest radical scavenging activity followed by Cycas thouarsii. D. spinulosum showed the highest protective activity against DNA damage which was higher than the positive control. D. spinulosum showed the highest antihemolytic antioxidant activity followed by Encephalartos laurentianus. D. spinulosum presented the highest antimicrobial activity among the tested plants. Most Cycadales tested plants especially D. spinulosum displayed significant biological activities that are promising for future drug discovery.