We present theory and experiments demonstrating the existence of invariant manifolds that impede the motion of microswimmers in two-dimensional fluid flows. One-way barriers are apparent in a hyperbolic fluid flow that block the swimming of both smooth-swimming and run-and-tumble emph{Bacillus subtilis} bacteria. We identify key phase-space structures, called swimming invariant manifolds (SwIMs), that serve as separatrices between different regions of long-time swimmer behavior. When projected into $xy$-space, the edges of the SwIMs act as one-way barriers, consistent with the experiments.
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