Aims: To devise and evaluate a strategy for isolating members of the Streptomyces violaceusniger phenotypic cluster, which are known to be a promising source of bioactive metabolites. Methods and Results: The treatment of four soil samples with 1.5% phenol (30degreesC, 30 min) prior to inoculation on humic acid-vitamin (HV) agar eliminated most of the streptomycetes and other bacterial populations. The surviving streptomycetes on the HV isolation plates were subcultured, and species-group identification was made according to the probabilistic identification system of Williams et al. (1989). Of the 133 streptomycetes subcultured, 102 (77%), were assigned to the S. violaceusniger cluster. A test with an overlay technique revealed that all of these S. violaceusniger-cluster isolates had broad antimicrobial spectra, as they inhibited the growth of all test Gram-positive bacteria, yeasts and filamentous fungi. Antitumour activity against colon carcinoma cells was found among 68 or 67%, of these S. violaceusniger-cluster isolates, following growth in submerged culture. Conclusions: Chemical pretreatment of soil samples with phenol reduces the growth of ubiquitous Streptomyces species, thereby facilitating the recovery of less-abundant S. violaceusniger-cluster strains that are characterized by high antimicrobial and antitumour activities. Significance and Impact of the Study: The development and application of new methodologies with which to selectively isolate rare, bioactive streptomycete groups is important for discovering novel secondary metabolites with bioactive properties.