The intense use of herbicides for weed control in agriculture causes selection pressure on soil microbiota and water ecosystems, possibly resulting in changes to microbial processes, such as biogeochemical cycles. These xenobiotics may increase the production of reactive oxygen species and consequently affect the survival of microorganisms, which need to develop strategies to adapt to these conditions and maintain their ecological functionality. This study analyzed the adaptive responses of bacterial isolates belonging to the same species, originating from two different environments (water and soil), and subjected to selection pressure by herbicides. The effects of herbicide Callisto and its active ingredient, mesotrione, induced different adaptation strategies on the cellular, enzymatic, and structural systems of two Bacillus megaterium isolates obtained from these environments. The lipid saturation patterns observed may have affected membrane permeability in response to this herbicide. Moreover, this may have led to different levels of responses involving superoxide dismutase and catalase activities, and enzyme polymorphisms. Due to these response systems, the strain isolated from water exhibited higher growth rates than did the soil strain, in evaluations made in oligotrophic culture media, which would be more like that found in semi-pristine aquatic environments. The influence of the intracellular oxidizing environments, which changed the mode of degradation of mesotrione in our experimental model and produced different metabolites, can also be observed in soil and water at sites related to agriculture. Since the different metabolites may present different levels of toxicity, we suggest that this fact should be considered in studies on the fate of agrochemicals in different environments.