Abstract Background The increasing threat of bacterial resistance promotes the need for antibiotic stewardship programs to foster responsible antimicrobial use. Therefore, guidelines for prudent use supported by an online stewardship tool (AntibioticScout.ch) were introduced in Switzerland in December 2016. They recommend (with decreasing preference) a first, second or third line antimicrobial for treatment. The objective of this study was to evaluate antimicrobial prescriptions for Swiss calves before (2016) and after (2018) the launch of these guidelines. Cases of calves with pneumonia, diarrhea and otitis from a university hospital and eight private practices in Switzerland were included. Data on anamnesis, clinical findings, diagnostic work-up and treatment were collected. Type and percentages [95% confidence interval] of antimicrobial prescriptions were compared between 2016 and 2018. Results Of the total number of calves, 88.2% [85.4–90.6] in 2016 (n = 625) and 88.4% [85.7–90.7] in 2018 (n = 655) were treated with antibiotics. The use of highest priority critically important antimicrobials (HPCIAs) decreased from 52.7% [48.6–56.9] in 2016 to 38.0% [34.2–41.9] in 2018; this decrease was found at the university hospital and in private practice and in cases with pneumonia and diarrhea. Particularly the use of fluoroquinolones decreased (2016: 43.1% [39.2–47.2]; 2018: 31.1% [27.6–34.8]). Overall, the number of first line treatments increased from 12.8% [10.4–15.6] in 2016 to 20.2% [17.3–23.4] in 2018. In cases of pneumonia, first line treatments increased (2016: 15.3% [11.6–19.9]; 2018: 26.5% [21.8–31.9]) and third line treatments decreased (2016: 43.5% [38.0–49.3]; 2018: 27.9% [23.1–33.3]); this was seen at the university hospital, whereas in private practice only a decrease of third line treatments was observed. In cases of diarrhea, more second line at the expense of unlisted antimicrobials were prescribed at the university hospital in 2018. Antimicrobial treatment of calves with otitis did not change from 2016 to 2018. Conclusions After the introduction of AntibioticScout.ch, more prudent use was observed in the treatment of calves with pneumonia and diarrhea as less HPCIAs, particularly fluoroquinolones, and more first line antimicrobials were prescribed. However, the overall frequency of antimicrobial treatment did not change and the use of HPCIAs was still common in 2018, especially in private practices. Therefore, further antimicrobial stewardship activities are necessary.