The search for new, powerful antimicrobials is essential to respond to the current worldwide spread of antibiotic-resistant pathogens. Sponge-associated bacteria have great potential for production of antimicrobials against resistant and multidrug resistant (MDR) pathogenic bacteria, but only few species of the Class Homoscleromorpha have been screened for these activities so far. The aim of this study was to isolate and identify sponge-associated bacteria active against antibiotic-resistant pathogens from sponges of classes Homoscleromorpha and Demospongiae. By employing five different growth conditions, a total of 239 colony-forming units were isolated and remained viable. Among these, 17 (7.1%) isolates presented antimicrobial activity against pathogenic and (multi)drug resistant bacteria including vancomycin-resistant Enterococcus faecalis, Escherichia coli, Citrobacter freundii, Klebsiella penumoniae, Staphylococcus spp. and Streptococcus spp. Bioactive bacteria belonging to genera Bacillus and Vibrio were identified at species level and the DNA fingerprint patterns showed that strains of the same genus were not clonally related. The most active strains belong to genus Bacillus and were isolated from Oscarella sp., Plakina cyanorosea and Chondrilla caribensis. Our results show for the first time that sponge-associated strains of Bacillus pumilus and Bacillus muralis have high anti-MDR activity, and that the Homoscleromorpha may be a better source of such anti-MDR active bacteria than the Demospongiae. These results suggest that marine bacteria associated to homoscleromorph sponges may be an interesting source of new antimicrobial substances with biotechnological potential to treat infections caused by antibiotic-resistant bacteria.