Background: Recently, there is a trend in the world to use natural products to attain wanted therapeutic effects and to avoid unwanted adverse effects. Probiotics are one of the most important of these natural products, which are friendly microbiota, can boost immunity and reduce inflammation and infection. Aims and Objectives: Therefore, we examined the protective effect of Nigella sativa (NS), gum arabic (GA), and Lactobacillus (LA) as a positive control against pathogenicity induced by Escherichia coli and Staphylococcus aureus infection in Wistar rats at microbiological and molecular levels. Materials and Methods: A total of 66 rats were used for this study and allocated into 11 groups. Rats were infected by either E. coli or S. aureus after administration of NS and/or GA for 2 weeks and continued for 7 days later. Another 2 groups infected by E. coli or S. aureus were administered LA as a positive control. Kidney, lung, and spleen were collected for the total bacterial count and molecular examination of immune cytokines and oxidative stress expression. Results: NS exerted a strongly antioxidant effect in E. coli and S. aureus infected rats as indicated by the upregulation in mRNA of superoxide dismutase (SOD) and glutathione peroxidase (GPX), whereas GA groups showed downregulation in SOD and GPX expression. NS normalized the inhibitory effect of GA when co-administered together. Immunomodulatory effect of NS and GA showed a clear response for Th-1 more than Th-2. NS significantly increased interferon-gamma (INF-γ) and inhibited interleukin-4 (IL-4) expression in E. coli infected group, while the combination of each gives the lowest results. In S. aureus infected rats, the NS extract upregulates the expression of IL-12 and GA downregulated IL-12 either alone or in combination with NS. The bacterial count per gram of organs significantly decreased in NS extract (P < 0.01), whereas increased significantly in GA and NS plus GA-treated groups compared to LA administered rats. Conclusion: NS possess strong and more antioxidant, immunomodulatory, and antibacterial properties than GA, which showed only an increase in Th-1 response INF-γ.