A cross-sectional study was performed on Nile tilapia (Oreochromis niloticus) in two ponds in the Qassim region of Saudi Arabia from October 2019 to March 2020. It was carried out to isolate and identify Gram-negative and Gram-positive bacteria from pond water and different fish organs, including blood, liver, and kidney samples. Blood, liver, and kidney functions were detected in this study. Bacterial isolates linked with fish could be transmitted to humans by eating fish or through the handling of fish that cause human diseases. Different bacterial isolates were identified depending on their growth properties and biochemical reactions in a specific culture medium. Escherichia coli, Salmonella typhi, Bacillus sp., Aeromonas hydrophila, Pseudomonas aeruginosa, Enterococcus faecalis, and Acinetobacter sp. was isolated from water of pond A and pond B. The most prevalent bacteria isolated from fish blood in ponds A and B were Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. The most prevalent bacteria isolated from the fish liver in ponds A and B was Escherichia coli. The most prevalent bacteria isolated from the fish kidneys in ponds A and B was Staphylococcus aureus. This study showed that bacterial infections in freshwater fish cause alterations in blood parameters such as RBCs, WBCs, Hb, Ht, and serum parameters of liver and kidney functions.