Background/aim: Because biofilms are resistant to antibiotics and biocides, they usually cause chronic persistent infections, which are arduous to cure and have high mortality and morbidity. Our study aimed to investigate the efficiency of orthophthalaldehyde, peracetic acid, hydrogen peroxide, and sodium hypochlorite on Pseudomonas aeruginosa and Staphylococcus aureus biofilm layers and live microbial cells. Materials and methods: Biofilm layers were determined by crystal violet assay and live microbial cells were determined using a resazurin assay. Results: For Pseudomonas aeruginosa, sodium hypochlorite showed the most influential disinfection because it diminished 83.6% of the biofilm layer and decreased 99.7% of live microbial cells. For Staphylococcus aureus, hydrogen peroxide was determined the most active disinfectant with 80.3% reduction of the biofilm layer. Sodium hypochlorite was also determined to be the most efficient disinfectant with 99.8% reduction of live microbial cells. Sodium hypochlorite was the most influential disinfectant on biofilm layers and live microbial cells of both microorganisms. Conclusion: We concluded that if we use sodium hypochlorite at a high level as a disinfectant for both surfaces and medical equipment, it is beneficial to prevent infections related to biofilms. More studies about prevention of biofilm occurrence and standardization of the methods for investigating disinfectants? effects are necessary.