Bovine mastitis (BM) is the most significant and prevalent disease in dairy cattle, causing significant financial losses worldwide. Among environmental causes of BM, Escherichia coli (E.coli) is considered a leading cause for sub-clinical, and clinical mastitis (CM) developing systemic reaction, abnormal milk changes, and udder inflammation. Therefore, the purpose of this study was to determine the prevalence rate of E.coli in dairy cows among cases of CM, assess the effectiveness of antibiotics using antimicrobial sensitivity testing, and to measure changes in biochemical markers within cows with CM as opposed to those that were treated. Out of the 64 milk samples used in the current investigation, eleven (17.1%) tested positive for E.coli. Antimicrobial sensitivity test results showed that E.coli strains are completely resistant to penicillin, tetracyclines, and beta-lactam antibiotics while being highly susceptible to the fluoroquinolone antibiotic class. The study revealed that the milk from cows with mastitis had significantly lower levels of total protein (TP), triglycerides (TG), calcium, and total antioxidant capacity (TAC) and significantly higher amounts of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), sodium chloride, and malondialdehyde (MDA) than the milk from non-mastitis and treated cows. From this study, authors concluded that the systemic administration of fluoroquinolones is very effective to NSAIDs in the treatment of CM cases that were infected with the E.coli pathogen. In addition, biochemical analysis of milk samples can be achieved to diagnose and confirm the potential role of treatment protocol in animal mastitis.