Abstract How does environmental complexity affect the evolution of single genes? Here, we measured the effects of a set of mutants of Bacillus subtilis glutamate dehydrogenase across 19 different environments - from homogenous single cell populations in liquid media to heterogeneous biofilms, plant roots and soil communities. The effects of individual gene mutations on organismal fitness were highly reproducible in liquid cultures. Strikingly, however, 84% of the tested alleles showed opposing fitness effects under different growth conditions (environmental pleiotropy). In biofilms and soil samples, different alleles dominated in parallel replica experiments. Accordingly, we found that in these heterogeneous cell communities the fate of mutations was dictated by a combination of selection and drift. The latter relates to programmed prophage excisions that occurred along biofilm development. Overall, per individual condition, by the combined action of selection, pleiotropy and chance, a wide range of glutamate dehydrogenase mutations persisted and sometimes fixated. However, across longer periods and multiple environments, nearly all this diversity would be lost - indeed, considering all environments and conditions we have tested, wild-type is the fittest allele.