Single crystal (−201) β-Ga2O3 substrates doped with Si and Sn have been thermally annealed in N2 and O2 atmospheres. Structural and electrical properties evaluation was performed via a number of experimental methods in order to quantify the effects of the doping and annealing ambient on the properties of these samples. All samples annealed in O2 exhibited significantly lower carrier concentration, as determined by capacitance–voltage measurements. Schottky barrier diodes exhibited excellent rectification when the Ga2O3 was annealed in N2, and significantly lower forward current using O2-annealed Ga2O3 substrates. Deep level transient spectroscopy revealed four deep trap levels with activation energies in the range of 0.40–1.07 eV. Electron spin resonance showed a decrease in shallow donor concentration, and cathodoluminescence spectroscopy revealed nearly two orders of magnitude lower emission intensity in O2-annealed Ga2O3 samples. Raman spectroscopy revealed a carrier concentration dependent Raman mode around 254 cm−1 observed only when the final anneal of (−201) β-Ga2O3 was not done in O2. Secondary ion mass spectroscopy measurements revealed diffusion of unintentional Fe towards the surface of the (−201) Ga2O3 samples after annealing in O2. Depth resolved positron annihilation spectroscopy showed an increased density of vacancy defects in the bulk region of O2-annealed Ga2O3 substrates.