The aim of this study was to measure changes in biochemical markers in the peripartum period of primiparous Holstein cows diagnosed with subclinical and clinical mastitis. In this study, 37 dairy cows were monitored daily during milking until 60 days postpartum and were categorized according to the occurrence of clinical mastitis (group mastitis (GM), n = 9) or subclinical mastitis (group subclinical mastitis (GSUB), n = 10) or absence of symptoms (control group (CG), n = 18). Blood samples were collected weekly from -30 to 60 days from calving. Samples were grouped for prepartum (-30 to 0 days from calving), early postpartum (0 to 30 days from calving), and late postpartum (30 to 60 days from calving) periods. Prepartum serum non-esterified fatty acid (NEFA) concentration was higher in GM than in CG (P < 0.01). In addition, CG had higher prepartum serum glucose concentration than GM (P = 0.03). In the early postpartum period, aspartate aminotransferase (AST) activity was lower in CG than in GSUB (P < 0.05), and in the late postpartum period, AST activity was lower in CG than GSUB and GM (P = 0.01). Somatic cell count was higher during the early and late postpartum periods for GM and GSUB when compared to CG (P < 0.01). In this study, primiparous cows with low glucose and higher NEFA in the prepartum were more susceptible for mastitis in the early postpartum, probably due to low immune function associated to a more negative energy balance. In sum, increased prepartum serum NEFA concentration and decreased glucose in primiparous cows were associated with clinical mastitis incidence in the postpartum period.