The understanding of the particle size and interparticle spatial effects on the electrical conductivity properties of molecularly mediated thin film assemblies of nanoparticles is important for exploring the unique electrical properties in applications such as chemical sensors and biosensors. This paper reports findings of an investigation of such effects for thin film assemblies of gold nanoparticles of highly-monodispersed sizes (2–4 nm) using alkyl dithiols of different chain lengths (0.8–2 nm). The conductivities of the thin films were measured using interdigitated microelectrodes as the platform. Experimental results have shown that the activation energy increases with chain length and decreases with particle size, and the electron tunneling decay term decreases with particle size. The results have revealed that the experimentally determined conductivity and activation energy data for the nanoparticle films quantitatively match the calculations from electrostatic model of granular metals through the electron tunneling mechanism. The strong correlation between the experimental and calculated data was attributed to a combination of the high monodispersity of the nanoparticles and the uniformity and stability of the thin film assemblies. These findings have provided an important set of systematic data supporting the applicability of the activated electron tunneling theory to the molecularly-mediated thin film assemblies of gold nanoparticles, which have important implications for the design and fine-tuning of nanostructured thin films as chemical and biological sensing materials.