An increasing proportion of the Earth's surface is illuminated at night. In aquatic ecosystems, artificial light at night (ALAN) may influence microbial communities living in the sediments. These communities are highly diverse and play an important role in the global carbon cycle. We combined field and laboratory experiments using sediments from an agricultural drainage system to examine how ALAN affects communities and alters carbon mineralization. Two identical light infrastructures were installed parallel to a drainage ditch before the start of the experiment. DNA metabarcoding indicated that both sediment communities were similar. After one was lit for five months (July–December 2012) we observed an increase in photoautotroph abundance (diatoms, Cyanobacteria ) in ALAN-exposed sediments. In laboratory incubations mimicking summer and winter (six weeks each), communities in sediments that were exposed to ALAN for 1 year (July 2012–June 2013) showed less overall seasonal change compared with ALAN-naive sediments. Nocturnal community respiration was reduced in ALAN-exposed sediments. In long-term exposed summer-sediments, we observed a shift from negative to positive net ecosystem production. Our results indicate ALAN may alter sediment microbial communities over time, with implications for ecosystem-level functions. It may thus have the potential to transform inland waters to nocturnal carbon sinks.