Positive species interactions underlie the functioning of ecosystems. Given their importance, it is crucial to understand the stability of positive interactions over evolutionary timescales, in both constant and fluctuating environments; e.g., environments interrupted with periods of competition. We addressed this question using a two-species microbial system in which we modulated interactions according to the nutrient provided. We evolved in parallel four experimental replicates of species growing in isolation or together in consortia for 200 generations in both a constant and fluctuating environment with daily changes between commensalism and competition. We sequenced full genomes of single clones isolated at different time points during the experiment. We found that the two species coexisted over 200 generations in the constant commensal environment. In contrast, in the fluctuating environment, coexistence broke down when one of the species went extinct in two out of four cases. We showed that extinction was highly deterministic: when we replayed the evolution experiment from an intermediate time point we repeatably reproduced species extinction. We further show that these dynamics were driven by adaptive mutations in a small set of genes. In conclusion, in a fluctuating environment, rapid evolution destabilizes the long-term stability of positive pairwise interactions.