Simple SummaryZucchini yellow mosaic virus (ZYMV) is one of the most prevalent plant viruses and represents a great challenge for crop production sustainability and human food supplementation. Conventional approaches to disease control depend on the heavy application of hazardous chemicals, which implies severe environmental, animal, and human health challenges. Using natural and microbial products represents a promising tool for sustainable and eco-friendly agricultural applications. The foliar application of the rhizobacteria Paenibacillus polymyxa strain SZYM revealed significant enhancements in squash growth parameters and enzyme production when compared to the non-treated plants. On the other hand, there was also a significant decrease in ZYMV accumulation, accompanied with a significant increase in the transcriptional levels of defense-related genes on plants inoculated with P. polymyxa. Additionally, a significant decrease in non-enzymatic oxidative stress markers was observed, as well as a considerable increase in reactive oxygen species scavenging enzymes. The present study showed that the P. polymyxa strain SZYM could be considered a promising rhizobacterium for enhancing plant growth and defense, and consequently a possible biocontrol agent of plant viral infections.The use of microbial products as natural biocontrol agents to increase a plant’s systemic resistance to viral infections is a promising way to make agriculture more sustainable and less harmful to the environment. The rhizobacterium Paenibacillus polymyxa has been shown to have strong biocontrol action against plant diseases, but its antiviral activity has been little investigated. Here, the efficiency of the culture filtrate of the P. polymyxa strain SZYM (Acc# ON149452) to protect squash (Cucurbita pepo L.) plants against a Zucchini yellow mosaic virus (ZYMV, Acc# ON159933) infection was evaluated. Under greenhouse conditions, the foliar application of the culture filtrate of SZYM either in protective or curative treatment conditions enhanced squash growth, reduced disease severity, and decreased ZYMV accumulation levels in the treated plants when compared to the non-treated plants. The protective treatment group exhibited the highest inhibitory effect (80%), with significant increases in their total soluble carbohydrates, total soluble protein content, ascorbic acid content, and free radical scavenging activity. Furthermore, a considerable increase in the activities of reactive oxygen species scavenging enzymes (superoxide dismutase, polyphenol oxidase, and peroxidase) were also found. In addition, the induction of systemic resistance with a significant elevation in the transcriptional levels of polyphenolic pathway genes (CHS, PAL, and C3H) and pathogenesis-related genes (PR-1 and PR-3) was observed. Out of the 14 detected compounds in the GC–MS analysis, propanoic acid, benzenedicarboxylic acid, tetradecanoic acid, and their derivatives, as well as pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) were the primary ingredient compounds in the ethyl acetate extract of the SZYM-culture filtrate. Such compounds may act as elicitor molecules that induce systemic resistance against viral infection. Consequently, P. polymyxa can be considered a powerful plant growth-promoting bacterium (PGPB) in agricultural applications as well as a source of bioactive compounds for sustainable disease management. As far as we know, this is the first time that P. polymyxa has been shown to fight viruses in plants.