Judicious antimicrobial stewardship in livestock industry is needed to reduce the use of antimicrobials (AMU) and the associated risk of antimicrobial resistance. Biosecurity measures are acknowledged for their role against the spread of diseases and the importance in reducing AMU in different species. However, their effectiveness in beef production has been scarcely considered. The aim of this study was to investigate the effect of the quarantine strategy on AMU in beef cattle. A total of 1206 Charolaise animals in five farms were included in the trial. Roughly half of the animals followed the standard procedure of the fattening cycle (no-quarantine; NO-QUA group) and half followed a 30-day period of quarantine (QUA group) since their arrival. Performance and antimicrobial data were recorded and a treatment incidence 100 (TI100it) per animal was calculated. Penicillins was the most used class of antimicrobials. Differences between groups were significant for males only, with NO-QUA group having greater TI100it (3.76 vs. 3.24; p < 0.05) and lower body weight at slaughter (713.4 vs. 723.7 kg; p < 0.05) than QUA group. Results suggest that quarantine strategy can reduce AMU in males without compromising their performance, whereas further investigation is needed for females.