The response to spring desiccation and autumn (rainfall-related) rewetting of sediment microbial functioning was investigated in a Mediterranean seasonal pond. Surface (0–2 cm depth) and subsurface (2–10 cm) sediments were sampled during the pond emptying, the dry period, and after a rain event, and characterized for potential microbial activities [substrate-induced respiration (SIR), denitrification enzyme activity (DEA), and nitrification enzyme activity (NEA)]. Eddy covariance fluxes of CO2 were also measured as an integrated value of the respiration of the sediment. Our results showed that short-term variations in the sediment water content can strongly affect the microbial functioning, whereas the metabolic structure of the microbial community was not strongly modified. The duration of the dry period influenced the intensity of microbial potentials and the surface layer responded differently from the subsurface layer, which was less affected by variations in water content. SIR and DEA were strongly stimulated during early summer desiccation of the sediment, while NEA was stimulated during late desiccation period. DEA and NEA potentials remained very high in sediment after 1 month of drought. Eddy covariance measurements confirmed that global CO2 fluxes were enhanced during early desiccation, decreased under dry conditions, and finally re-enhanced after a rainfall. These results suggest that management of such ponds can influence CO2 and N2O emissions, and that some precautions in the management (limitation of dry-wet alternations and duration of emptying) could help reduce greenhouse gas emissions. Copyright © 2010 John Wiley & Sons, Ltd.