Global change has led to the increased duration and frequency of droughts and may affect the microbial-mediated biochemical processes of intermittent rivers and ephemeral streams (IRES). Effects of flow desiccation on the physical structure and community structure of benthic biofilms of IRES have been addressed, however the dynamic responses of biofilm functions related to ecosystem processes during the dry-wet transition remain poorly understood. Herein, dynamic changes in biofilm metabolic activities were investigated during short-term (25-day) and long-term (90-day) desiccation, both followed by a 20-day rewetting period. Distinct response patterns of biofilm metabolism were observed based on flow conditions. Specifically, biofilms were completely desiccated after 10 days of drying. Biofilm ecosystem metabolism, represented by the ratio of gross primary production (GPP) and community respiration (CR), was significantly inhibited during desiccation and gradually recovered back to autotrophic after rewetting due to the high resilience of GPP. Also, the potential metabolic activities of biofilms were maintained during desiccation and showed a tendency to recover after rewetting. While long-term desiccation caused irreparable damage to the total carbon metabolism of biofilms that could not be recovered to the control level even after 20 days of rewetting. Moreover, the metabolic activities of amine and amino acids showed an inconsistent pattern of recovery with total carbon metabolism, indicating the development of selective carbon metabolism. This research provides direct evidence that the increased non-flow periods affects biofilm-mediated carbon biogeochemical processes, which should be taken into consideration for the decision-making of the ecological and environmental flow of IRES.