Not all agricultural practices are sustainable; however, non-thermal plasma treatment of seeds may be an eco-friendly alternative to improve macroscopic plant growth parameters. Despite the numerous successful results of plasma-seed treatments reported in the literature, there is a large gap in our understanding of how non-thermal plasma treatments affect seeds, especially due to the plethora of physical, chemical, and biological variables. This study uses RNA sequencing to characterize the changes in gene transcription in Arabidopsis thaliana (L.) Heynh. seeds 6 days after exposure to surface dielectric barrier discharge plasma treatment. Here, we provide an overview of all pathways that are differentially expressed where few genes are upregulated and many genes are downregulated. Our results reveal that plasma treatment time is a parameter that can activate different pathways in plant defense. An 80 s treatment upregulates the glucosinolate pathway, a defense response to insects and herbivores to deter feeding, whereas a shorter treatment of 60 s upregulates the phenylpropanoid pathway, which reinforces the cell wall with lignin and produces antimicrobial compounds, a defense response to bacterial or fungal plant pathogens. It seems that plasma elicits a wounding response from the seed in addition to redox changes. This suggests that plasma treatment can be potentially applied in agriculture to protect plants against abiotic and biotic stresses without discharging residues into the environment.