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 (800mg×4) or 10days (1000mg×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 7-9days 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.