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Ortho-phthalaldehyde (OPA) was tested against a range of organisms including glutaraldehyde-resistant mycobacteria, Bacillus subtilis spores and coat-defective spores. Glutaraldehyde (GTA) and peracetic acid (PAA) were tested for comparative purposes. Both suspension and carrier tests were performed using a range of concentrations and exposure times. All three biocides were very effective (≥ 5 log reduction) against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in suspension tests. OPA and GTA (PAA was not tested) were also very effective against Staph. aureus and Ps. aeruginosa in carrier tests. OPA showed good activity against the mycobacteria tested including the two GTA-resistant strains, but 0·5% w/v OPA was found not to be sporicidal. However, limited activity was found with higher concentrations and pH values. Coat-defective spores were more susceptible to OPA, suggesting that the coat may be responsible for this resistance. The findings of this study suggest that OPA is effective against GTA-resistant mycobacteria and that it is a viable alternative to GTA for high level disinfection.