Solithromycin (CEM-101) is a "fourth-generation" macrolide, as it has three binding site and is acid stable. The three binding sites confer activity against bacteria resistant to the older macrolides and ketolides, including multidrug-resistant Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi). The objective of this study was to evaluate solithromycin pharmacokinetics (PK), middle ear fluid (MEF) concentrations, and microbiologic efficacy in a chinchilla model of experimental otitis media (EOM) due to strains of S. pneumoniae or NTHi. Plasma PK (maximum concentration of drug in serum [Cmax] and area under the concentration-time curve from 0 to 24 h [AUC0-24]) and middle ear fluid (MEF) concentrations were determined. Isolates with specified antimicrobial susceptibility patterns were inoculated directly into the middle ear (ME). Plasma and MEF were collected for PK and MEF cultures performed to determine efficacy. Solithromycin administered at 150 mg/kg of body weight/day resulted in Cmax and AUC0-24 values of 2.2 µg/ml and 27.4 µg · h/ml in plasma and 1.7 µg/ml and 28.2 µg · h/ml in extracellular MEF on day 1. By day 3, Cmax and AUC0-24 values had increased to 4.5 µg/ml and 54 µg · h/ml in plasma and 4.8 µg/ml and 98.6 µg · h/ml in extracellular MEF. For NTHi EOM, three isolates with MIC/minimal bactericidal concentration (MBC) ratios of 0.5/1 µg/ml (isolate BCH1), 2/2 µg/ml (isolate BMC1247C), and 4/4 µg/ml (isolate BMC1213C) were selected. The MEF of >85% of animals infected with BCH1 and BMC1247C was sterilized. For NTHi BMC1213, >85% of MEF cultures remained positive. For S. pneumoniae EOM, 3 isolates with MIC/MBC ratios of 0.06/0.125 µg/ml (S. pneumoniae 331), 0.125/1 µg/ml (S. pneumoniae CP-645 [MLSB phenotype]), and 0.5/2 µg/ml (CP-712 [mefA subclass mefA resistance]) were selected. Solithromycin sterilized MEF in 100% of animals infected with S. pneumoniae 331 and S. pneumoniae CP-645. ME infection persisted in 60% of animals infected with CP-712. In a model of EOM, solithromycin sterilized MEF in >85% of animals challenged with NTHi with an MIC of ≤2 µg/ml and 100% of ME infected with S. pneumoniae with an MIC of ≤0.125 µg/ml.