The antibiotic compound diacetylphloroglucinol (DAPG) is produced by various isolates of the beneficial bacterium Pseudomonas fluorescens. DAPG can be toxic to plants, bacteria, fungi and viruses, and DAPG production has been associated with activity against some plant pathogens. This study was undertaken to investigate toxicity of DAPG to certain soil-dwelling nematodes. The study included the following free-living and plant-parasitic nematodes: Caenorhabditis elegans, Heterodera glycines, Meloidogyne incognita, Pratylenchus scribneri, Pristionchus pacificus, Rhabditis rainai and Xiphinema americanum. Eggs, juveniles and/or adults of these nematodes were exposed to synthetic DAPG: DAPG concentrations ranged from 1 μg/ml to 75 or 100 μg/ml. Percentages of egg hatch and of mobile vermiform stages were recorded. Egg hatch of M. incognita was inhibited by DAPG; mobility of second-stage juveniles was not affected. Conversely, egg hatch of C. elegans was stimulated by DAPG, at least during early hours of exposure. However, as with M. incognita, juvenile (J1) mobility was not altered, nor was mobility of adults. DAPG was toxic to adults of X. americanum. The other four nematode species did not respond to DAPG (based on the measured parameters). The data indicate that DAPG production by soil-dwelling bacteria would not directly result in suppression of population numbers of every plant-pathogenic or bacterial-feeding nematode species. Augmentation of DAPG-producing P. fluorescens populations for biological control of nematodes should be targeted to pathogenic nematode species that have been shown to be sensitive to the compound, or used for indirect effects, such as induced systemic resistance.