Soil microbiome and multi-trophic relationships are essential for the stability and functioning of agroecosystems. However, little is known about how farming systems and alternative methods for controlling plant pathogens modulate microbial communities, soil mesofauna and plant productivity. In this study, we assessed the composition of eukaryotic microbial groups using a high-throughput sequencing approach (18S rRNA gene marker), the populations of parasitic and free-living nematodes, plant productivity and their inter-relationships in long-term conventional and organic farming systems. The diversity of the fungal community increased in the organic farming system compared to the conventional farming system, whereas the diversity of the protist community was similar between the two farming systems. Compared to conventional farming, organic farming increased the population of free-living nematodes and suppressed plant parasitic nematodes belonging to Meloidogynidae and Pratylenchidae. Fungal diversity and community structure appeared to be related to nematode suppression in the system receiving organic fertilizer, which was characterized by component microbial groups known to be involved in the suppression of soil pathogens. Unraveling the microbiome and multi-trophic interactions in different farming systems may permit the management of the soil environment toward more sustainable control of plant pathogens.