Staphylococcal mastitis (SM) is a frequent disease in the dairy cattle that is costly to treat. This study aimed to investigate the alterations in the levels of procalcitonin (PCT), neopterin (NPT), haptoglobin (HP), serum amyloid A (SAA), proinflammatory cytokines (IL-1β, IL-8, TNF-α, IF-γ) and oxidative stress (OS) biomarkers in Holstein dairy cows with SM under field conditions. In addition, we also evaluated the role of examined biomarkers in disease pathogenesis and their use as diagnostic biomarkers for the disease in dairy cows. Fifty-three dairy cows with SM, including those with infections caused by Staphylococcus aureus (n = 42) and methicillin resistant S. aureus (MRSA) (n = 11) were selected for this study. In addition, 20 healthy dairy cows were enrolled as a control group. Higher serum levels of PCT, NP, IL-1β, IL-8, TNF-α, IF-γ, HP and SAA and a state of OS was detected in SM group in comparison with the controls. Moreover, the levels of all examined biomarkers in mastitic cows with S. aureus when compared with those infected with MRSA was not significantly different. All examined biomarkers demonstrated a significant degree of discrimination between SM cows and healthy controls (the area under the curve (AUC) ranged from 83.6 for SAA to 100 for PCT). Our study showed that SM in dairy cows was associated with substantial changes in serum PCT, NPT, Acute phase proteins (APPs), proinflammatory cytokines, and OS levels. This study demonstrates that clinical examination in tandem with quantification of PCT, NPT, APPs and cytokines, OS biomarkers could be a useful assessment tool for SM in dairy cows.