Antibiotic resistance of strains of Staphylococcus aureus isolated from bovine milk is of concern internationally. The objective of this study was to investigate trends of resistance of S. aureus to antibiotics administered to dairy cows in 19 South African and one Zambian dairy herds (participating in the South African proactive udder health management programme) and to identify possible contributing factors. The resistance of S. aureus strains to eight commonly used antibiotics in South Africa from 2001 to 2010 was evaluated. Staphylococcus aureus isolates (n = 2532) were selected from cows with subclinical mastitis in 20 herds routinely sampled as part of the proactive udder health management programme. The isolates were selected from milk samples that had somatic cell counts more than 400 000 cells/mL and were tested for antibiotic resistance using a standard Kirby–Bauer test with published clinical breakpoints. The prevalence of antibiotic resistance was evaluated as a percentage of S. aureus isolates susceptible out of the total numbers for each antibiotic selected per year. Staphylococcus aureus showed a significant increase in percentage of susceptible isolates over time for all antibiotics tested except for ampicillin. The overall prevalence of mastitis did not change during the study period. However, the prevalence of mastitis caused by S. aureus (mostly subclinical cases) in the selected herds decreased numerically but not significantly. Reduction in the incidence of antibiotic resistance shown by S. aureus was presumed to be a result of the application of the proactive udder health management programme. The fact that the overall prevalence of mastitis was kept stable was possibly because of the influence of the management programme in conjunction with the return of infections caused by non-resistant strains.