Omadacycline is a broad-spectrum aminomethylcycline approved in October 2018 by the U.S. Food and Drug Administration for treating acute bacterial skin and skin structure infections and community-acquired pneumonia as both an oral and intravenous once-daily formulation. In this report, the activities of omadacycline and comparators were tested against 49,000 nonduplicate bacterial isolates collected prospectively during 2016 to 2018 from medical centers in Europe (24,500 isolates, 40 medical centers [19 countries]) and the United States (24,500 isolates, 33 medical centers [23 states and all 9 U.S. census divisions]). Omadacycline was tested by broth microdilution following the methods in Clinical and Laboratory Standards Institute document M07 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 11th ed., 2018). Omadacycline (MIC50/90, 0.12/0.25 mg/liter) inhibited 98.6% of Staphylococcus aureus isolates at ≤0.5 mg/liter, including 96.3% of methicillin-resistant S. aureus isolates and 99.8% of methicillin-susceptible S. aureus isolates. Omadacycline potency was comparable for Streptococcus pneumoniae (MIC50/90, 0.06/0.12 mg/liter), viridans group streptococci (MIC50/90, 0.06/0.12 mg/liter), and beta-hemolytic streptococci (MIC50/90, 0.12/0.25 mg/liter), regardless of species and susceptibility to penicillin, macrolides, or tetracycline. Omadacycline was active against all Enterobacterales tested (MIC50/90, 1/8 mg/liter; 87.5% of isolates were inhibited at ≤4 mg/liter) except Proteus mirabilis (MIC50/90, 16/>32 mg/liter) and indole-positive Proteus spp. (MIC50/90, 8/32 mg/liter) and was most active against Escherichia coli (MIC50/90, 0.5/2 mg/liter), Klebsiella oxytoca (MIC50/90, 1/2 mg/liter), and Citrobacter spp. (MIC50/90, 1/4 mg/liter). Omadacycline inhibited 92.4% of Enterobacter cloacae species complex and 88.5% of Klebsiella pneumoniae isolates at ≤4 mg/liter. Omadacycline was active against Haemophilus influenzae (MIC50/90, 0.5/1 mg/liter), regardless of β-lactamase status, and against Moraxella catarrhalis (MIC50/90, ≤0.12/0.25 mg/liter). The potent activity of omadacycline against Gram-positive and -negative bacteria indicates that omadacycline merits further study in serious infections in which multidrug resistance and mixed Gram-positive and Gram-negative bacterial infections may be a concern.