Aquaponic systems are an integrated way to produce fish and plants together with mutual benefits. Fish provide nutrients to plants on the one side, and plant nutrients uptake allow water reuse for fish on the other side. In this kind of system, the use of phytosanitary treatments to control plant pathogens is sensitive because of the risk of toxicity for fish present in the same water loop, especially coupled aquaponics. Among plant pathogens, Pythium aphanidermatum is a most problematic microorganism due to the Oomycete’s capacity to produce mobile form of dispersion (zoospores) in the recirculated water. Therefore, this study aimed at elucidating the potential antagonistic capacity of aquaponic water against P. aphanidermatum diseases. It was shown that aquaponic water presented an inhibitory effect on P. aphanidermatum mycelial growth in in vitro conditions. The same result was observed when lettuce plants growing in aquaponic water were inoculated by the same plant pathogen. Aquaponic lettuce was then compared to lettuce grown in hydroponic water or complemented aquaponic water (aquaponic water plus mineral nutrients). The disease was suppressed in the presence of aquaponic water, contrary to lettuce grown in hydroponic water or complemented aquaponic water. Root microbiota were analyzed by 16S rDNA and ITS Illumina sequencing to determine the cause of this aquaponic suppressive action. It was determined that the diversity and the composition of the root microbiota were significantly correlated with the suppressive effect of aquaponic water. Several taxa identified by metabarcoding were suspected to be involved in this effect. Moreover, few of these microorganisms, at the genus level, are known to have an antagonistic effect against P. aphanidermatum. These innovative results indicate that aquaponic water could be an interesting and novel source of antagonistic agents adapted to control P. aphanidermatum diseases in soilless culture.