CEM-101 is a novel fluoroketolide with reported high potency against diverse groups of Gram-positive (Micrococcus spp., viridans group streptococci, Corynebacterium spp. Listeria monocytogenes, Clostridium spp., etc.) and Gram-negative bacteria (Neisseria gonorrhoeae, Campylobacter jejuni, Helicobacter pylori, Bacteroides fragilis, Shigella spp., etc.), including mycoplasma and ureaplasma, as well as bacteria commonly associated with community-acquired respiratory tract infections and skin and skin structure infections. In this study, CEM-101 and comparator antimicrobials were tested against a collection of very low prevalence aerobic and anaerobic bacteria collected via the SENTRY Antimicrobial Surveillance Program platform. CEM-101 was highly active against all Gram-positive organisms (MIC 50 , 0.015 μg/mL) as compared with telithromycin (MIC 50 , 0.06 μg/mL), clarithromycin (MIC 50 , 0.12 μg/mL), and erythromycin (MIC 50 , 0.25 μg/mL). Among Gram-negative pathogens, CEM-101 also displayed a high potency against most strains (MIC 50 , 4 μg/mL) but was found to be equivalent or less active when compared with other antimicrobials tested with MIC 50 values ranging from ≤0.12 μg/mL for levofloxacin to 8 μg/mL for telithromycin. Among the strict anaerobic species, CEM-101 activity mirrored that of the aerobic species: high activity against the Gram-positive anaerobes (MIC 50 results ranging from ≤0.03 μg/mL to 0.12 μg/mL) and equivalent or less susceptible against Gram-negative anaerobes. Our in vitro antimicrobial susceptibility results for CEM-101 demonstrate better activity compared with other MLS B class agents among a diverse group of uncommonly isolated bacterial pathogens; these results provide an impetus for possible expanded indications during Phase 2 and 3 clinical trials.