Nigella sativa (NS), as a traditional medical plant, is widely used in Middle Eastern countries and Indonesia. Nigella sativa extract exhibits a wide range of characteristics that are beneficial to human health, including anti-inflammatory, antidiabetic, antihypertensive, antifungal, antiviral, antimicrobial, and antioxidant properties. Thymoquinone (TQ) is the major active compound of NS extract, which has potent antioxidant and anti-inflammatory effects in both in vitro and in vivo model studies. Considering the results of existing studies, the researchers were interested in conducting a study on the effect of administration of Nigella Sativa Extract (ENS) on the growth of Streptococcus pneumoniae colonies in Wistar rats’ model of bacterial conjunctivitis. This study is an analytical study with a true experimental design with a pretest-posttest control group design approach, using a wistar rat model of bacterial conjunctivitis as the object of research. The population of this study were all male Wistar rats aged 4 to 9 weeks. This research was conducted in the laboratory of the Faculty of Pharmacy and Medicine, University of North Sumatra and took place for approximately 3 months, from May 2022 to July 2022. The study sample was 25 male rats divided into 5 groups. One way ANOVA test showed that there was a significant difference in the number of Streptococcus pneumoniae colonies based on the treatment groups (p<0.001). The Posthoc further test showed a significant difference in the number of Streptococcus pneumoniae between the groups of rats given 20% Habbatus Sauda (p=0.034) and ofloxacin (p=0.026). Nigella sativa extract demonstrated antibacterial activity against Streptococcus pneumonia. This suggests that Nigella sativa carries great potential as an effective antimicrobial agent for medicinal purposes, especially in bacterial conjunctivitis.