Biofouling is an important factor threatening the safe and efficient service of marine engineering materials, so it is of great significance to develop efficient, environmentally friendly and low-cost antifouling materials. In this paper, two kinds of α-MoO3 powder materials were prepared by one-step thermal decomposition at 450 °C and 750 °C, designated as S450 and S750. And the samples were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and infrared spectroscopy (FTIR). It indicated that the α-MoO3 obtained at 450 °C and 750 °C showed nanoscale plate-like structure and micron-sized long plate-like structure, respectively. Plate counting was used to evaluate the antimicrobial effects of two materials on Pseudomonas aeruginosa (P. aeruginosa) in the absence of light. The results showed that the antibacterial rates of S450 and S750 at concentrations of 0.25, 0.5, 0.75, and 1 mg/ml were 27.3%, 99%, 100%, 100% and 16.1%, 30.1%, 52.3%, 73.6%, respectively. It indicated that both α-MoO3 obtained under the two conditions showed good antibacterial properties in the absence of light. The pH value of the solution decreases with the increase of the α-MoO3 concentration, and the concentration of Mo ions increases with the increase of the α-MoO3 concentration. The antibacterial activity of α-MoO3 in the absence of light stems from the synergistic effect of dissolution of H3O+ and Mo ions. Compared to S750, S450 exhibits higher antibacterial rate, which may be due to the higher solubility of S450, resulting in more H3O+ and Mo ions release.