Heavy metals are not biodegradable and are deposited, incorporated, and bioaccumulated in aquatic organisms. The Nile tilapia, Oreochromis niloticus, is regarded as one of the most significant biomonitors in the aquatic ecosystem for detecting heavy metal contamination. During the current study, concentrations of heavy metals, markers of oxidative stress and inflammatory responses were assessed in the liver, kidney, and muscle tissues of O. niloticus fish collected from five key points in the Damietta Branch (El-kanater El-Khayriya, Benha, Zefta, Talkha, and El-Serw) in addition to undergoing their histopathological examination. Furthermore, native electrophoretic proteins and the relative gene expressions of antioxidant enzymes and inflammatory markers in the target tissues were assayed. The tilapia fish obtained from El-Serw had the highest quantities of heavy metals in their liver and muscle, followed by Talkha, Zefta, and Benha. When comparing the liver and muscle of tilapia fish collected from El-Serw, Talkha, Zefta, and Benha to those collected from E. EL-Khayria, measurements of the antioxidant system (both enzymatic and non-enzymatic) significantly decreased (P≤0.05) in tandem with rising levels of inflammatory markers. All of the tilapia fish's tissues that were taken from El-Serw had significant histopathological changes. Furthermore, the tissues of fish taken from El-Serw exhibited the lowest similarity percentages when compared to those from E. EL-Khayria, according to the electrophoretic protein patterns. According to the molecular assays, O. niloticus fish collected from El-Serw, Talkha, Zefta, and Benha had significantly lower relative gene expressions of antioxidant enzymes (P≤0.05) in conjunction with higher levels of inflammatory markers than fish collected from E. El-Khayria. The study revealed that severe histopathological, physiological, and molecular alterations were noticed in the target tissues of O. niloticus fish gathered from El-Serw with those gathered from other areas.