This study investigated the in vitro efficacy of emamectin benzoate and glyphosate against monogeneans from Colossoma macropomum and tolerance of this species to these therapeutic drugs. In vitro assays demonstrated that concentrations of emamectin benzoate (12.5, 16.6, 20.8 and 25.0g/L) and glyphosate (2,000; 3,000; 4,000 and 5,000mg/L) were 100% effective against Anacanthorus spathulatus, Notozothecium janauachensis and Mymarothecium boegeri. The highest concentrations of emamectin benzoate (20.8 and 25.0g/L) were 100% effective after 30 minutes of exposure. For glyphosate, the highest concentrations were the most effective, and 4,000 and 5,000 mg/L immobilized 100% of parasites in 45 minutes and 30 minutes, respectively. Scanning electron microscopy revealed that the parasites exposed to 20.8 and 25.0g/L of emamectin benzoate and 5,000 mg/L of glyphosate presented integument covered with deeper wrinkles. Behavioral changes occurred in C. macropomum exposed to all concentrations of both drugs, were as mortality occurred following exposure to emamectin benzoate and glyphosate at 2.0,12,5 and 25.0g/L and 500, 1,000, 2,000, 3,000, 4,000 and 5,000mg/L, respectively. Low concentrations of emamectin benzoate and of glyphosate were tolerated by C. macropomum, but these concentrations may have not efficacy against infection of monogeneans.