Cytochrome P450 1A (CYP1A), a cytochrome P450 gene, participates in the metabolism of xenobiotics. In this research, levels of CYP1A expression were evaluated in treated and untreated bacterial infections in Oreochromis niloticus organs and histopathological changes in splenic tissues. Twelve groups of O. niloticus fish: control (T_1-4), fish infected with Aeromonas hydrophila (T₅-₈), and fish infected with Pseudomonas fluorescens (T₉-₁₂) groups) were treated with Nigella sativa oil and ciprofloxacin. In the uninfected group, treatment doses did not affect CYP1A expression levels. In infected fish, the level of CYP1A expression significantly increased without treatment while relative CYP1A expression was significantly downregulated when treated with N. sativa oil and ciprofloxacin additives. The combined treatments had a synergetic effect on the downregulation of CYP1A expression in the liver and gill. The main histological alterations in the spleen of infected fish were severe hyperplasia of melanomacrophage centers (MMCs), which was higher in the A. hydrophila infected fish than in the P. fluorescens infected fish. The results clarify the mechanisms of CYP1A gene expression and MMCs against pathogens and their consequences.