Herbicide consumption is yearly increasing in order to enhance crop yield and its quality. However, these chemical compounds are threatening environment, and for instance, soil. Thus, tendency for natural bioherbicide use is increasing. In fact, these “bio-compounds” are supposed less harmful for the environment and its biodiversity. However, the evaluation of the environmental impact for these compounds is still not widely studied. This is because of multiple limitations in classical evaluation protocols (e.g. half-life). Thus, new omics-based methods are being developed in order to adapt new suitable protocols and assess the environmental risk of such biocontrol products. Recently, an innovative metabolomics-based approach was developed in the laboratory as an alternative tool for the evaluation of the environmental impact. This approach named “Environmental Metabolic Footprinting” (EMF), was applied on pure chemical pesticides in soil [1], and then on a complex bio-insecticide in sediments [2], using Liquid Chromatography-Mass Spectrometry (LC-MS). The aim of this approach is to determine the “resilience time” as a new integrative evaluation concept. It corresponds to the time required for the dissipation of the compound and its effect on the matrix microbiome. In fact, this approach takes in consideration the analysis of the whole “meta-metabolome” of the environmental matrix. This meta-metabolome contains the pesticide and its by-products (xenometabolome), but also the microbial metabolome. Thus, as EMF approach proved efficiency and several advantages, improving this approach was considered by introducing High Resolution Mass Spectrometry (HRMS), using a Quadrupole/Time-of-Flight Mass Spectrometer (QToF MS). This improvement allowed higher selectivity by exact mass measuring. Hence, potential bias issued from interferences were eliminated. In addition, necessary information for biomarkers discovery and identification were provided. These advantages are necessary to study complex natural biopesticides containing several unknown metabolites. Thus, this work was applied to evaluate the environmental impact of a natural complex bioherbicide; the Myrica gale extract, containing the “Myrigalone A”. The impact of this natural bioherbicide will be compared to the pure synthetized Myrigalone A compound. A reference product, “Sulcotrione”, a well-known β-triketone herbicide, will be included in the study. This bioherbicide presenting high affinity to the soil, makes the kinetics monitoring insufficient for low concentration abundance. Thus, the interest of applying EMF is to analyze its by-products and the impact on microbial metabolome. For that, a 150 days kinetic experiment was performed with 10 time points in soil microcosms. Five replicates were done for each treatment and time condition. After soil extraction, the extracts were analyzed after optimizing an exhaustive analytical method by LC-QToF system. Acquired data were processed on “Workflow4metabolomics” online platform after critical review and rigorous optimization of preprocessing parameters. Processed data were used for multivariate analysis, and resilience time was explored by comparison of herbicide-spiked microcosms with controlled microcosms. Results showed high discrimination between controlled batch and the Myrica gale extract batch. Hence, biomarkers “fishing” and discovery is planned in order to identify discriminant metabolites, so we can explain better the environmental impact of this bioherbicide. [1]: Patil et al. 2016. Sci. Total Environ. 566–567:552–558 [2]: Salvia et al. 2018. Environ. Sci. Pollut. Res. 25(30):29841–29847