Single-electron transistors incorporating single ∼1 nm gold nanocluster (AuNCs) and pentacene as a complex charge transport system have been used to study the quantum Coulomb blockade and its single electron tunnelling behaviour at room temperature (RT) (300 K). Monodisperse ultra-small (0.86 ± 0.30 nm) AuNCs were deposited by the tilted-target sputtering technique into 12 nm nanogaps fabricated by high-resolution e-beam lithography. Tunnelling resistance was modulated to ∼10(9) Ω by addition of a pentacene layer, allowing clear observation of quantum staircases and Coulomb oscillations with on/off current modulation ratio of ∼100 in RT current-voltage measurements. The electron addition energy and average quantized energy level spacing were found to be 282 and 80.4 meV, respectively, which are significantly larger than the thermal energy at 300 K (25.9 meV).