We tested the antimicrobial activity of orthophthalaldehyde (OPA) against 21 strains (16 species) of pathogenic microorganisms that cause hospital-associated infections. Changes in hepatitis B surface antigen (HBs-Ag) resulting from the addition of OPA to HBs-Ag-positive serum were measured using a radioimmunoassay. We also examined the effect of immersing medical instruments in OPA (0.55%) for 168 h at room temperature. OPA (0.5%, 0.37% and 0.25%) killed 11 strains of vegetative bacteria within 15 s, and it killed the test micro-organisms faster than 3.0% glutaraldehyde (GTA). Incubation with OPA or GTA caused levels of HBs-Ag to fall below a cut-off value within 30 s. OPA did not adversely affect instruments made from various materials. OPA demonstrated more effective antimicrobial activity than GTA against a range of microorganisms. We conclude that OPA should replace GTA as the first-choice high-level disinfectant for endoscopes, considering its antimicrobial efficacy and low inhalation toxicity.