Introduction: Rhodococcus equi is a facultative intracellular pathogen that causes severe pneumonia in foals. The most common treatment today is rifampicin in combination with clarithromycin or azithromycin. In 2020, EMA classified rifampicin in the category of antibiotics that should be avoided in veterinary medicine and its use in foals could be questioned. The aim of this study was therefore to evaluate the in vitro efficacy of these antibiotics as well as that of possible alternatives under conditions as close as possible to those found in the foal. Methods: The activity of rifampicin, clarithromycine, azithromycine, gamithromycine, doxycycline and marbofloxacine alone or in combination were assessed by MIC determination, checkerboard assays and time-kill curves on R. equi ATCC 33701. Two parameters were taken into account to mimic intra- and extracellular conditions: pH of the medium and maximum antibiotic concentrations in the epithelial ling fluid and in macrophages of foals reported in literature. Results: At intracellular pH of 5.8, the MICs of the macrolides, gamithromycin, azithromycin and clarithromycin were 32–1000 times higher than at extracellular pH of 7.5. For rifampicin, marbofloxacin and doxycycline, the influence of the pH was minimal on the MIC. Checkerboards assays, performed at different pH, showed no clear synergy between macrolides and rifampicin. In time-kill curves, the bacterial killing was quite slow for monotherapies and there was also no clear advantage of the combinations. The lower activity of macrolides at acidic pH was not compensated by the high concentrations reached after accumulation in the cells. As an example, bacterial counts with gamithromycin at 10 μg/mL, did not differ from control at pH 5.8 while it can eradicate bacteria at 5 μg/mL at pH 7.5. Some combinations of doxycycline or marbofloxacin with macrolides also appear to be quite effective without the use of rifampicin. Conclusion: This study showed that macrolides are less effective at acidic pH, and that the reference treatment (rifampicin + macrolides) shows a slightly better bactericidal activity compared to monotherapy under extracellular conditions only. However, even if combinations do not have a great interest in increasing bactericidal activity, they may nevertheless limit resistance selection. Since clinical trials are particularly costly and risky for foals, taking into account conditions encountered in vivo in in vitro studies will allow for the best prediction of expected effects in foals.