Molecular transport in nanofluidic systems exhibits properties that are unique to the nanoscale. Here, the electrostatic and steric interactions between particle and surfaces become dominant in determining particle transport. At the solid–liquid interface of charged surfaces an electric double layer (EDL) forms due to electrostatic interactions between surfaces and charged particles. In these systems, tunable charge-selective nanochannels can be generated by manipulating electrostatic gating via co-ions exclusion and counterions enrichment of the EDL at the solid–liquid interface. In this context, electrostatic gating has been used to modulate the selectivity of nanofluidic membranes for drug delivery, nanofluidic transistors, and FlowFET, among other applications. While an extensive body of literature investigating nanofluidic systems exists, there is a lack of a comprehensive analysis accounting for all major parameters involved in these systems. Here we performed an all-encompassing modeling investigation corroborated by experimental analysis to assess the influence of nanochannel size, electrolyte properties, surface chemistry, gate voltage, dielectric properties, and molecular charge and size on the exclusion and enrichment of charged analytes in nanochannels. We found that the leakage current in electrostatic gating, often overlooked, plays a dominant role in molecular exclusion. Importantly, by independently considering all ionic species, we found that counterions compete for EDL formation at the surface proximity, resulting in concentration distributions that are nearly impossible to predict with analytical models. Achieving a deeper understanding of these nanofluidic phenomena will help the development of innovative miniaturized systems for both medical and industrial applications.