Cellular membranes are constantly bombarded with biomolecules and nanoscale particles, and cell functionality depends on the fraction of the bound/internalized entities. Understanding the biophysical parameters underlying this complex process is very difficult in live cells. Model membranes provide an ideal platform to obtain insight into the minimal and essential parameters involved in determining cell membrane-nanoparticle (NP) interaction. Here we report spontaneous binding and unbinding of semiconductor NPs, carrying different net charges and interacting with model biomembranes, using in situ neutron reflectivity (NR) and fluorescence microscopy studies. We observe a critical concentration of NPs above which they spontaneously unbind along with lipids from lipid monolayer membranes, leaving behind fewer bound NPs. This critical concentration varies depending on whether the NPs carry a net charge or are neutral, and is also governed by the extent of NP crowding for a fixed NP charge. The observations suggest a subtle interplay between electrostatics, membrane fluidity, and NP crowding effects, which eventually determines the adsorbed concentration for unbinding transition. Our study provides valuable microscopic insight into the parameters that could determine the biophysical process underlying NP uptake and ejection by cells which, in turn, can be utilized for their potential applications in bioimaging and drug delivery.