Cefquinome is a 4th generation cephalosporin that is approved in Brazil to treat Streptococcus agalactiae subclinical mastitis (SM). Nonetheless, cefquinome is critically important for humans and its exposure to dairy cows could be reduced if traditional drugs, such as cloxacillin, were still effective to treat S. agalactiae SM. The objectives of this randomized clinical trial were to: 1) estimate the cure rate of S. agalactiae SM treated with intramammary cloxacillin (CLOXIMM), intramammary cefquinome (CEFIMM), or intramuscular cefquinome (CEFIM), as compared with a negative control group (CON); and 2) test the hypothesis that CLOXIMM is non-inferior to CEFIMM to treat S. agalactiae SM. Seven farms were visited for screening and milk samples were collected from all quarters of all lactating cows for microbiological examination. Streptococcus agalactiae-positive cows were randomized into 4 groups: CLOXIMM (n = 60), CEFIMM (n = 64), CEFIM (n = 31), and CON (n = 16 quarters). Treatments were administered per label directions (once a day for 3 d for CLOXIMM and CEFIM, and every 12 h for 3 consecutive milkings for CEFIMM). Microbiological cure was assessed at 14 (D14) and 21 (D21) days after beginning of treatments. Outcomes were bacteriological cure at D14 (CURE14), D21 (CURE21), and D14 and D21 (CURE1421). Logistic regression was used to compare the cure rate between each treatment and CON. Non-inferiority analysis was performed considering a one-sided 95% confidence interval (CI) and a non-inferiority margin of 0.15. The cure rate for CLOXIMM, CEFIMM, CEFIM, and CON was 88, 100, 55, and 19% for CURE14; 85, 97, 52, and 7% for CURE21; and 85, 97, 52, and 6% for CURE1421, respectively (P < 0.05 for the comparisons between each treatment and CON). Although the cure rate difference between CLOXIMM and CEFIMM (0.12; 95% CI: 0.04–0.20) at D14 was < 0.15, non-inferiority was inconclusive because the required sample size has not been reached. Preliminary results suggest that cloxacillin can still be used to treat S. agalactiae SM, instead of drugs that should be prioritized for human use.