We contribute to the knowledge of charophyte meadows as key components of aquatic systems by analysing how they affect wetland sediments. We performed a factorial-design experiment with limnocorrals (outdoor mesocosms) in a Mediterranean protected wetland with presence or absence of charophytes [Chara vulgaris (CV) and Chara hispida (CH), planted from cultures or recruited in situ from germination of their fructifications]. The first 1 cm-surficial and 2 cm-bottom sediment layers were analysed for cladoceran ephippia, ostracods valves, benthic community of bacteria and periphytic biofilm, and charophyte fructifications. In the surficial sediment, the ephippia density was fourfold higher in the conditions with charophytes than in sites with no-charophytes and higher apparent viability was found. The surficial sediment periphyton biofilm was composed mainly of diatoms, with tenfold higher biomass underneath charophytes, and a much diverse community. The specific microhabitat generated by each charophyte species was reflected in the different abundances and relationships between the analysed components, firstly establishing a divergence with the sediment without meadows and, secondly, a distinction between the meadows of CH and CV that exhibit particular morphology-architecture, might exudate different metabolites and might have different allelopathic capacities over microalgae and microinvertebrates. Thus, the charophyte–sediment tandem is relevant for biodiversity and habitat conservation.