Eurasian watermilfoil (Myriophyllum spicatum L.) is an invasive aquatic plant that can hybridize with the native northern watermilfoil (M. sibiricum Kom.). These milfoil hybrids (M. spicatum × M. sibiricum) are becoming more prevalent in many lakes where the invasive and the native milfoil co-occur. Hybrid plants are more vigorous than either parent with a faster growth rate and lower sensitivity to some herbicides. The aquatic herbicides endothall and 2,4-D provide two effective modes of action for management of the hybrids. For more than a decade, these two herbicides have been used in combination as an effective control option and a resistance management strategy. How this combination impacts herbicide movement and efficacy is unknown. Therefore, the objective of this research was to determine the activity of endothall and 2,4-D combined compared with activity applied alone. Absorption and translocation of endothall, 2,4-D, and the combination was determined in hybrid plants over a 96-h time course. Endothall accumulation was not impacted when these herbicides were applied in combination; however, 2,4-D accumulation increased by 80%, relative to when 2,4-D was applied alone. Endothall translocation from shoots to roots decreased by almost 50% when applied in combination with 2,4-D (alone = 16.7% ± 2.6%; combination = 9.2% ± 1.2%). Shoot-to-root translocation of 2,4-D also decreased when the two herbicides were applied in combination (24.8% ± 2.6% when applied alone to only 3.93% ± 0.4% when in the presence of endothall). This research demonstrates that combining herbicides can significantly impact herbicide activity in plants. Future research is needed to determine whether this reduced translocation negatively impacts operational effectiveness when these herbicides are applied in combination.