Objectives Physicians must leverage several factors when making antibiotic therapy decisions, including route of administration and duration of therapy. Oral administration provides several potential advantages including increased accessibility, prevention of hospitalizations and earlier discharges. Sulopenem—a broad-spectrum, synthetic penem β-lactam agent—uniquely possesses both oral and IV formulations along with noted stability among antimicrobial-resistant subsets. This study evaluated the in vitro activity of sulopenem and comparator agents against contemporary Enterobacterales and anaerobic clinical isolates predominantly from patients with bloodstream, intra-abdominal and urinary tract infections. Methods A contemporary collection of 1647 Enterobacterales and 559 anaerobic isolates was assembled from medical centres in Europe and the USA. Isolates were susceptibility tested using the CLSI reference methods: broth microdilution for Enterobacterales and agar dilution for anaerobes. Results Sulopenem demonstrated potent in vitro antimicrobial activity (MIC50/90, 0.03/0.25 mg/L) against Enterobacterales isolates regardless of infection type, inhibiting 99.2% of isolates at ≤1 mg/L. This activity was conserved against resistant phenotypes including ESBL-phenotype Escherichia coli (MIC50/90, 0.03/0.06 mg/L) and ESBL-phenotype Klebsiella pneumoniae (MIC50/90, 0.06/1 mg/L). Sulopenem maintained activity against ciprofloxacin-, nitrofurantoin- and trimethoprim/sulfamethoxazole-non-susceptible subsets (MIC50/90, 0.03–0.06/0.12–0.5 mg/L). Against anaerobic isolates, sulopenem (98.9% inhibited at ≤4 mg/L) and meropenem [98.4% susceptible (CLSI)] were the most active compounds tested. Conclusions The potent in vitro activity of sulopenem against this large collection of recent Enterobacterales and anaerobic clinical isolates from multiple infection types supports its further clinical evaluation in the treatment of intra-abdominal and urinary tract infections.