Bacteria produce extracellular enzymes to obtain resources from complex chemical substrates, but this strategy is vulnerable to cheating by cells that take up reaction products without paying the cost of enzyme production. We hypothesized that cheating would suppress enzyme production in co-cultures of cheater and producer bacteria, particularly under well-mixed conditions. To test this hypothesis, we monitored protease expression and frequencies of Pseudomonas fluorescens producer and cheater genotypes over time in mixed liquid cultures and on agar plates. In mixed culture inoculated with equal frequencies of cheaters and producers, enzyme concentration declined to zero after 20 days, consistent with our hypothesis.