Glyphosate, a systemic herbicide, is used against weeds in agricultural fields as well as against invasive plants in pastures, forest plantations, and urban environments. Its frequent and widespread use can negatively impact the surrounding natural non-target vegetation following the accidental drift of spray droplets, leaching, or persistence as residues in the soil. Because ferns possess a life cycle with independent sporophytic and gametophytic generations, herbicides may cause a different impact on each life stage. The objective of the present study was to evaluate the effect of four concentrations of glyphosate (0.33, 0.65, 2.72, and 10.89 g active ingredient L^-1) and water as control treatments on spores, gametophytes, and two sporophyte size classes of one native (Blechnum appendiculatum Willd.) and two introduced fern species in Mexico (Macrothelypteris torresiana (Gaudich.) Ching and Thelypteris dentata (Forssk.) E.P. St.John). Spore germination was evaluated 10 days after herbicide treatment and the percentage of remaining green tissue was measured at 7, 30, and 90 days after herbicide treatment. Plant survival was determined at the end of the experiment. Glyphosate suppressed spore germination nearly completely and increased tissue discoloration of all green life stages at higher concentrations and after longer time intervals. After 7 and 30 days, small sporophytes of all three species were significantly more discolored than gametophytes and large sporophytes at concentrations ≤ 0.65 g a.i. L^-1, although after 90 days differences among life stages were no longer significant, and no life stage survived at concentrations ≥ 2.72 g a.i. L^-1. At the lowest concentration (0.33 g a.i. L^-1), however, 50–69% of the plants of all three species and life stages survived after 90 days post-treatment. Macrothelypteris torresiana was significantly more tolerant to glyphosate than the other two species during the first 30 days after treatment, mainly because of its less damaged small sporophytes, perhaps due to a thicker waxy cuticle that may initially reduce herbicide absorption in this species. We conclude that even glyphosate concentrations of 0.33 g a.i. L^-1 may negatively impact natural spore banks of ferns and result in mortality of 31–50% of all green life stages. Such negative effects may also eliminate at least the most susceptible fern species in habitats that are frequently exposed to such glyphosate concentrations.