Cooperation should be favored under environmental conditions allowing the preferential interaction of cooperators among themselves and limiting interactions with defectors. Bacteria cooperating to kill competitors by secreting a toxin evolved during several hundred generations in two environments: a viscous environment that should promote cooperator assortment, and a nonviscous environment that should not allow such preferential interaction. A quantitative decrease in cooperation was observed in all populations, but as expected, cooperation was maintained at a higher level in the viscous environment. Mutants that are resistant against but not producing the toxin were identified at a low frequency in a few populations from the viscous environment and at a high frequency in all the populations from the nonviscous environment. The underlying mutations were identified. Relative fitness of the cooperator and mutant genotypes were obtained with bacteria that were isogenic, except for the identified mutations. Competition experiments indicated that cooperation is not favored by environmental viscosity as imposed in our system and suggested that when it comes to cooperation, environmental viscosity should be considered not only in terms of individual movement, but also in terms of the distribution of the public good.