Pulmonary infections caused byMycobacterium abscessus(MAB) have been increasing in incidence in recent years, leading to chronic and many times fatal infections due to MAB’s natural resistance to most available antimicrobials. The use of bacteriophages (phages) in clinics is emerging as a novel treatment strategy to save the lives of patients suffering from drug-resistant, chronic, and disseminated infections. The substantial research indicates that phage–antibiotic combination therapy can display synergy and be clinically more effective than phage therapy alone. However, there is limited knowledge in the understanding of the molecular mechanisms in phage–mycobacteria interaction and the synergism of phage–antibiotic combinations. We generated the lytic mycobacteriophage library and studied phage specificity and the host range in MAB clinical isolates and characterized the phage’s ability to lyse the pathogen under various environmental and mammalian host stress conditions. Our results indicate that phage lytic efficiency is altered by environmental conditions, especially in conditions of biofilm and intracellular states of MAB. By utilizing the MAB gene knockout mutants of the MAB_0937c/MmpL10 drug efflux pump and MAB_0939/pks polyketide synthase enzyme, we discovered the surface glycolipid diacyltrehalose/polyacyltrehalose (DAT/PAT) as one of the major primary phage receptors in mycobacteria. We also established a set of phages that alter the MmpL10 multidrug efflux pump function in MAB through an evolutionary trade-off mechanism. The combination of these phages with antibiotics significantly decreases the number of viable bacteria when compared to phage or antibiotic-alone treatments. This study deepens our understanding of phage–mycobacteria interaction mechanisms and identifies therapeutic phages that can lower bacterial fitness by impairing an antibiotic efflux function and attenuating the MAB intrinsic resistance mechanism via targeted therapy.