Traditional antimicrobial antibiotics are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. The antibiotic era is threatened by the ruthless rise of resistance in bacterial infections. A significant role in these resistance profiles is attributed to multidrug efflux pumps. Hence, much effort is being directed towards developing new compounds to overcome this problem. During our screening program of efflux pumps inhibitors (EPI) produced by bioactive Moroccan Actinobacteria, 210 isolates were screened for their antibacterial activities against Escherichia coli strains containing a system of efflux pump AcrAB-TolC, fully functional, and its mutant, inactivated due to the insertion of transposon Tn903 in AcrAB operon, using the method of agar disc diffusion. The results showed that 14 isolates were able to produce EPI as they were active against the wild type strain but not against the mutant in comparison with the synthetic inhibitor L-Phe-L-Arg-β-naphthylamide (PaβN). We focused on the highest EPI activity produced by four strains (Z332, Z35/G, Z385/b and 136). Taxonomic studies and the 16S rDNA sequence indicated that these strains belonged to the Streptomyces species. This work could contribute to the discovery of a new class of antibacterial agents that could expand the therapeutic arsenal.