Household microorganisms mostly reside in the form of biofilms on wet surfaces in the bathroom and kitchen areas. Microorganisms constituting biofilm communities are less susceptible than their planktonic counterparts to antimicrobial agents and are thus difficult to control. Traditionally, chemical disinfectants have been used as cleaners in the household environment. Recently, incorporation of ozone as a microbial control agent in the consumer product industry has been initiated as an alternative method of disinfection. In this study, antimicrobial efficacy of ozone and a commercial chlorinated disinfectant was evaluated against the biofilm of a commonly occurring Pseudomonas aeruginosa and Klebsiella michiganensis, a newly described species recovered from toothbrush holder. Single species biofilm was grown on borosilicate glass and polycarbonate coupons using the Centers for Disease Control and Prevention (CDC) biofilm reactor. This is the first study demonstrating the biofilm forming capability of K. michiganensis under laboratory conditions using the CDC biofilm reactor. Planktonic cells and mature biofilms were exposed to ozonated water for 2 and 4 min and chlorinated cleaner for 2 min and 10 min. Based on the ozone stability study, the exposure experiments were carried out in two different ways. The type 1 treatment was carried out according to ASTM method E2871-12, with 5 mL of ozonated water as disinfectant in a 50 mL conical centrifuge tube; whereas in type 2 treatment, 14.0 mL of ozonated water was used to avoid any headspace in the capped culture tube. In biofilms, type 1 ozone treatment demonstrated an average log reduction of 0.88 (±0.13) and 0.12 (±0.01) for K. michiganensis and P. aeruginosa, respectively, in 2 min. Similarly, for type 2 treatment, the values were 1.56 (±0.58) and 0.27 (±0.08). In planktonic cells, log reduction was 2.61 (±0.11) in P. aeruginosa and > 4.0 in K. michiganensis, within 2 min. Chlorinated cleaner demonstrated > 4.0 log reduction in all cases. The disinfection efficacy of ozone type 1 treatment varied between bacterial species (p < 0.001) and exposure times (p < 0.04), but not the coupon types (p > 0.95). Within biofilms, log reduction varied significantly between ozone and chorine treatments (p < 0.001). Significant differences in log reduction were also observed between planktonic cells and biofilms (p < 0.001), target organisms (p < 0.05) and the types of ozone treatment (p < 0.05, for K michiganensis only). From the results, it can be concluded that K. michiganensis was more susceptible to the disinfectants compared to P. aeruginosa. Within a shorter contact time, commercial chlorinated cleaner was more effective against both biofilms and planktonic cells than ozonated water. © 2014, Springer-Verlag Berlin Heidelberg and the University of Milan.