Antibiotics are widely used in the bacterial treatment of animals and humans, which negatively affect agricultural activities. The present study evaluated the effect of enrofloxacin residues' presence in the soil from irrigation water contaminated with this antibiotic on the alfalfa (Medicago sativa) biomass. The study was conducted in a controlled plant growth chamber for 40 days, where 12 flowerpots were distributed in 3 treatments with three repetitions and three controls. Plants were watered with 100 mL of type 2 water during the first 16 days and with 48 h frequency, followed by 200 mL of water contaminated with enrofloxacin with 2, 20, and 200 μg L−1 with four days frequency until the harvest on day 40. Our results showed that the presence of enrofloxacin in the soil reduced carbon fixation in stems and leaves of alfalfa by 6.14% when irrigation was with 200 μg L−1, compared to the control group; likewise, the number of nodules was reduced by 91% when the concentration was 200 μg L−1, and the root length is reduced by 32% compared to the treatment average.