Herbicides are widely used in agricultural practices for preventing the proliferation of weeds. Upon reaching soil and water, herbicides can harm nontarget organisms, such as bacteria, which need an efficient defense mechanism to tolerate stress induced by herbicides. 2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide that exerts increased oxidative stress among bacterial communities. Bacterial isolates were obtained from the biofilm of tanks containing washing water from the packaging of different pesticides, including 2,4-D. The Pseudomonas sp. CMA-7.3 was selected because of its tolerance against 2,4-D toxicity, among several sensitive isolates from the biofilm collection. This study aimed to evaluate the antioxidative response system of the selected strain to 2,4-D. It was analyzed the activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase GPX enzymes, that are poorly known in the literature for bacterial systems. The Pseudomonas sp. CMA-7.3 presented an efficient response system in balancing the production of hydrogen peroxide, even at 25x the dose of 2,4-D used in agriculture. The antioxidative system was composed of Fe-SOD enzymes, less common than Mn-SOD in bacteria, and through the activities of KatA and KatB isoforms, working together with APX and GPX, having their activities coordinated possibly by quorum sensing molecules. The peroxide control is poorly documented for bacteria, and this work is unprecedented for Pseudomonas and 2,4-D. Not all bacteria harbor efficient response system to herbicides, therefore they could affect the diversity and functionality of microbiome in contaminated soils, thereby impacting agricultural production, environment sustainability and human health.