Extended therapy is an alternative approach to treat nonresponsive staphylococcal mastitis, although it has been used mainly in adult cows. This study investigated the efficacy of intramammary ceftiofur in the treatment of staphylococcal subclinical mastitis in primiparous cows. Sixty primiparous cows Holstein were divided into four groups (G1 = Group negative to staphylococci infection, G2 = Group positive to staphylococci infection untreated with intramammary ceftiofur, Group, G3 = Group treated with intramammary ceftiofur early lactation, G4 = Group treated with intramammary ceftiofur late lactation group) with milk samples collected at five moments (M0 = diagnosis of subclinical mastitis, M1 = 7 days after diagnosis of subclinical mastitis and early extended therapy, M2 = 14 after the diagnosis of subclinical mastitis, M3 = 21 days after diagnosis of mastitis) and M4 = 28 days after diagnosis of mastitis). In groups G3 and G4, which were treated with intramammary ceftiofur, the microbiological cure was evidenced by a reduction in SCC of 73.3% and 46.7%, respectively. Thus, in the treated groups (G3 + G4) and untreated (G2), cure rates were 60% and 26.7%, respectively (P <0.05). Forty-five strains of staphylococci were isolated, with a predominance of S. aureus (51,1%) and S. intermedius (48,8%). The in vitro antimicrobial susceptibility pattern showed higher sensitivity indices of isolates for oxacillin (80%), ceftiofur (77.8%), cephalexin (77.8%), ciprofloxacin (66.7%), and gentamicin (60%). In contrast, cloxacillin (24.4%), penicillin (35.6%), and tetracycline (44.5%) were ineffective antimicrobials for staphylococci isolates. Among the milk samples taken when M0 and M1 (7 days after isolation), 13.3% were positive for the detection of the inhibiting substances (antimicrobials) in Delvotest™ and all negative in the Snap test™, whereas in the M2 (14 days after isolation), 55% were positive in Delvotest™ and 46.7% in the Snap test™. Twenty-one days after diagnosis (M3), 16.7% revealed positive results in Delvotest™ and 11.7% in the Snap test™. For M4 (28 days after isolation), 11.7% showed positive results in Delvotest™, and 8.3% revealed positive results in the Snap test™. There was a reduction in SCC among all cured animals. Significant results were identified for milk production in the M0, so G1 produced more milk than the other three groups, G2, G3, and G4. A statistical association was observed between the reduction in the volume of daily milk and production in animals with subclinical mastitis, denoting losses in milk production in primiparous bovines infected during lactation by staphylococci. Extended intramammary therapy with ceftiofur in bovine primiparous cows subclinically affected by staphylococcal mastitis is effective mainly in cases treated at the beginning of lactation, showing microbiological cure and reduction in SCC.