Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV-infected population. These seemingly harmless non-pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV-associated complications. Although immune-evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome-enriched fractions fromMycobacterium bovisBacillus Calmette–Guérin (BCG) mono-infected and HIV–M. bovisBCG co-infected THP-1 cells by LC-MALDI-MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co-infection helped the survival of non-pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co-infection up-regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co-infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria,Mycobacterium avium,Mycobacterium kansasiiandMycobacterium phleithat exhibited increased survival during co-infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.