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Background: The intestinal microbiota functions as a reservoir of antibiotic resistance., Objectives: To evaluate penicillin V (phenoxymethylpenicillin) effects on the faecal microbiota with focus on beta-lactam resistance., Methods: We included 31 primary care patients with group A streptococcal pharyngotonsillitis treated with penicillin V for 5 (800 mg × 4) or 10 days (1000 mg × 3). Twenty-nine patients contributed with three faecal swab samples each. The faecal specimens were collected at the start of penicillin V treatment, after the last dose and at follow-up 7-9 days after completed treatment. Samples were inoculated semiquantitatively on selective screening agar plates to study beta-lactam resistance, species shifts among Enterobacterales and enterococci, and colonization with Candida spp. and Clostridioides difficile . Representative colonies were identified using MALDI-TOF. Results were analysed by non-parametric statistical methods., Results: An increase in the proportion of patients colonized with ampicillin-resistant Enterobacterales, from 52% to 86% ( P  = 0.007), and Enterobacterales with decreased susceptibility to third-generation cephalosporins, from 32% to 52% ( P  = 0.034), was observed between the first and second samples. This increase was no longer significant at follow-up. New colonization with ampicillin-resistant Enterobacterales species and non-Enterobacterales Gram-negative species was observed, and persisted at follow-up., Conclusions: Following treatment with penicillin V, we observed decreased susceptibility to ampicillin and third-generation cephalosporins, and prolonged colonization with non- Escherichia coli Gram-negative species. These findings challenge the perception that penicillin V has limited ecological effect on the intestinal microbiota, and emphasizes the importance of avoiding even narrow-spectrum antimicrobials when possible., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)