Although coagulase-negative staphylococci are the primary aetiological agents of subclinical mastitis in ewes, there is little information regarding vaccination against that infection. The objective of this study was to evaluate the efficacy of a vaccine against staphylococcal mastitis in ewes under experimental conditions. The antigen in the vaccine is based on a bacterin of Staphylococcus aureus strain, expressing the exopolysaccharide poly-N-acetylglucosamine (PNAG), which is involved in biofilm formation by these bacteria. Ewes in groups A (n = 17) or B (n = 6) were given an initial vaccination 5 weeks before expected lambing, followed by a repeat administration 21 days later. Ewes in groups C (n = 8) or D (n = 6) were unvaccinated controls. Ewes in group A (n = 17) or C (n = 8) were challenged with a biofilm-forming S. chromogenes; animals in subgroups A1 or C1 were challenged on the 10th and those in A2 or C2 on the 50th day after lambing. Ewes in groups B or D were uninoculated controls. Clinical examinations of ewes, ultrasonographic examinations of udder, milk yield measurements, blood sampling for detection of anti-PNAG specific antibodies and milk sample collection for bacteriological and cytological examinations were performed up to 52nd day post-challenge. Finally, biopsies were performed for mammary tissue collection for histopathological examination. Among group A ewes, 29% developed systemic signs and 59% signs in the inoculated gland; the respective figures for group C were 50% and 100% (P =  0.040 for mammary signs). The median total clinical score was 2.0 for A and 5.5 for C ewes (P =  0.025). For A, but not for C, clinical scores decreased progressively during the study (P =  0.018 and P =  0.47, respectively). The duration of mastitis was shorter in A (4 days) than in C (17.5 days) ewes (P =  0.022). Bacterial counts were lower in milk samples from A than from C ewes, for samples collected from the inoculated and the uninoculated (P <  0.01) mammary glands of these ewes. Somatic cell counts in samples from inoculated and uninoculated mammary glands of A ewes were higher than in samples of C ewes (P <  0.02). There were differences for gray-scale evaluations during ultrasonographic examination and for milk yield measurements between groups (P <  0.01). Median bacterial counts in tissue samples from A ewes (0 cfu g) were lower than in ones from C (6.5 cfu g) ewes (P =  0.041). The median score for histopathological findings in tissue samples from inoculated glands of A was lower than that for C ewes: 1 versus 2 (P =  0.014). It is concluded that mastitis was less severe in vaccinated animals, as indicated by a wide array of measures.