Mycobacterium abscessus lung infection is a major health problem for cystic fibrosis (CF) patients. Understanding the in vivo factors that influence the outcome of therapy may help addressing the poor correlation between in vitro and in vivo antibiotic efficacy. We evaluated the influence of interspecies interactions and lung epithelial cells on antibiotic efficacy. Therefore, single and dual-species biofilms of M. abscessus and a major CF pathogen (Pseudomonas aeruginosa) were cultured on a plastic surface or on in vivo-like three-dimensional (3-D) lung epithelial cells, and the activity of antibiotics (colistin, amikacin, clarithromycin, ceftazidime) in inhibiting biofilm formation was evaluated. Using the most physiologically relevant model (dual-species biofilms on 3-D cells), we observed that treatment with antibiotics during biofilm development inhibited P. aeruginosa but not M. abscessus biofilms, resulting in a competitive advantage for the latter. Clarithromycin efficacy against P. aeruginosa was inhibited by 3-D lung cells. In addition, biofilm induction of M. abscessus was observed by certain antibiotics on plastic but not on 3-D cells. Pseudomonas aeruginosa influenced the efficacy of certain antibiotics against M. abscessus, but not vice versa. In conclusion, these results suggest a role of host cells and interspecies interactions in bacterial responses to antimicrobials.