Abstract The great biodiversity of neotropical fish species that have external fertilization as a reproductive strategy, like the tambaqui, requires more careful analyzes in toxicological tests of the various pesticides implemented in Brazilian agriculture over the last few years. In this context, the objective of the present work was to evaluate possible sperm alterations in tambaqui (Colossoma macropomum) semen exposed to two different pesticide residues. Seminal samples of sexually mature tambaqui males from a local fish farm were used. Semen was collected eight hours after hormone induction into graduated glass tubes. After initial assessment of the lack of prior activation, the experiment was carried out in a factorial scheme, testing two pesticides widely used in agricultural systems (glyphosate and fenitrothion). For each pesticide, five concentrations were tested (6, 12, 24, 120 and 240 mg/L), with motility analysis at times 0, 30 and 60 seconds after activation. As a control, activation with 0.9% NaCl solution and motility analysis at the same times described for pesticides were used. Results indicate that in natura samples exhibited initial motility of 89.2 ± 4.9% and mean duration of 100 seconds (up to 10% sperm motility). The reduction in sperm motility occurred significantly (p < 0.05) after 30 seconds in all concentrations tested, except for the concentration of 240 mg/L because no activation was observed. The tests described here demonstrate that tambaqui semen was sensitive to the process of exposure to pesticide residues, and can be used in biomonitoring analyzes of the aforementioned agricultural pesticides.