The sea lamprey (Petromyzon marinus L.) is an invasive parasitic fish in the Laurentian Great Lakes that relies heavily on pheromones to synchronize migration and reproduction. Sexually mature male sea lamprey release a potent sex pheromone known as 3-keto petromyzonol sulfate (3kPZS), which robustly attracts mature females to spawning grounds. Given the dependency on sex pheromones for successful sea lamprey spawning, we sought to exploit the pheromone induced responses of 3kPZS to facilitate an effective and environmentally benign control strategy. We hypothesized that antagonists similar to 3kPZS will inhibit 3kPZS olfactory detection and disrupt behavioral responses of female sea lamprey. Candidate antagonists were identified by computationally screening millions of commercially available compounds as analogs of 3kPZS that match a set of key chemical functional groups and docked well into the receptor structure. More than 300 candidate antagonists were tested via electro-olfactogram for their ability to block olfactory responses to 3kPZS. Seven compounds blocked at least 50% of the olfactory response to 3kPZS. The 3-hydroxyl analog of 3kPZS, petromyzonol sulfate (PZS), blocked 92% of the olfactory response to 3kPZS. The top identified blockers of 3kPZS olfaction were tested in a bifurcated maze and then a stream bioassay to determine their ability to block behavioral responses to 3kPZS. When a mixture of PZS and 3kPZS was applied to the in stream treatment nest, the proportion of mature females that moved upstream and entered the treatment nest significantly decreased to 19%, compared to 52% entering the nest when 3kPZS was applied alone. This integrative approach has resulted in the identification of several antagonists, including a natural molecule that blocks pheromone reception in vivo at picomolar concentration. PZS has the potential utility to be implemented as a pheromone behavioral antagonist to control the invasive sea lamprey in the Great Lakes.