This study was carried out on a neglected component of wetlands: the picoplankton community. We analyzed the picoplanktonic community patterns and their related environmental factors in a hypertrophic semi-arid wetland located in Central Spain (Las Tablas de Daimiel National Park, TDNP). We determined the bacterial and autotrophic picoplankton (APP) abundance over a three-year period (1996: the end of a long drought period and 1997–1998: after flooding) in five sites of the wetland. The overall range of bacterial abundance was 0.2 × 10⁶ to 10 × 10⁶ cells/ml. The annual mean abundance increased in the wettest 1997. APP was composed mainly by coccoid phycocyanin-containing cyanobacteria, with the greatest abundance up to 25 × 10⁵ cells/ml. The annual mean also increased considerably in wetter 1997–98. Despite the large APP biomass in some sites, its percentage of total phytoplankton biomass was low (the annual average did not exceed 1.5%). We observed spatial heterogeneity in the picoplankton fraction depending on the fluctuating hydrology: bacteria tends to spatial homogeneity after flooding while APP showed only similarity among the output sites. Among the considered predictive variables (temperature, phosphorus, nitrogen, zooplankton, phytoplankton) of the picoplanktonic dynamics, temperature was the most closely correlated to picoplankton, especially to bacterial abundance. Further, in two factorial, coupled-hierarchical laboratory experiments (constant temperature), we searched for control mechanisms of picoplankton. We tested (a) the trophic cascade hypothesis by analyzing the effect of presence/absence of mosquitofish (experiment 1) or directly modifying the zooplanktonic community (experiment 2) and (b) the bottom-up regulation by altering the nutrient conditions (presence/absence of sediment in experiment 1; reducing the nutrient content in experiment 2). Bacterioplankton failed to show any behavior related to trophic cascade direct effects, while nutrients increased its abundance. APP was affected positively by nutrients and negatively by zooplankton grazing.