One of the core clinical criteria of Dementia with Lewy bodies (DLB) are fluctuations of cognition. Underlying processes may be rather functional than neurodegenerative, reflected by, for example, factors present in cerebrospinal fluid (CSF). The aim of this study was to identify in-vitro neuronal network activity (ivNNA) changes of CSF from DLB patients compared with patients with Parkinson’s disease (PD) and controls. Primary neuronal mouse cultures were grown on microelectrode arrays to record ivNNA when exposed to respective CSF samples. If exposed to CSF of DLB patients, ivNNA showed a reduced spike rate and burst rate compared with CSF of PD patients and controls. Our data are suggestive of the presence of functional factors in the CSF of DLB patients that differentiate network activity from PD patients and controls. Future studies should evaluate whether this pilot observation might be related to fluctuations of cognition in DLB.