Arbuscular mycorrhizal (AM) fungi can reduce the incidence and importance of plant root diseases caused by pathogens. The mechanisms involved are not well characterized. We used an in vitro experimental system to test the hypothesis that the extraradical mycelium of AM fungi can interfere directly with microorganisms in the mycosphere and directly or indirectly reduce the population of plant pathogens. This system permitted the isolation of soluble substances released by the extraradical mycelium of Glomus intraradices. The AM fungus was grown on Daucus carota transformed roots in one compartment, while only the extraradical mycelium was allowed to grow in a second compartment. A freezing and centrifugation technique was developed for the extraction and concentration of substances present in the compartment containing only the AM fungal mycelium. Four soil-inhabiting microorganisms were selected, and conidial germination (fungi) or growth (bacteria) of these was studied in the presence and absence (control) of the extract. In comparison with the control, the results indicated that both the growth of Pseudomonas chlororaphis and the conidial germination of Trichoderma harzianum were stimulated in the presence of the AM fungal extract. In contrast, conidial germination of Fusarium oxysporum f. sp. chrysanthemi was reduced while the growth of Clavibacter michiganensis subsp. michiganensis was not affected. The measured effects in general were directly correlated with extract concentration. Differences in pH were noted between the extract containing substances released by the AM fungus and the non-AM control, but no significant influence of the pH on growth or conidial germination was noted, confirming that substances released by the AM fungus in the growth medium is the main factor explaining differential growth of the microorganisms tested. The results suggest that direct interactions exist between AM fungi and soil microorganisms, which might lead to changes in microbial equilibrium detrimental to pathogens.