The use of personal protective equipment (PPE) has been considered the most effective way to avoid the contamination of healthcare workers by different microorganisms, including SARS-CoV-2. A spray disinfection technology (chamber) was developed, and its efficacy in instant decontamination of previously contaminated surfaces was evaluated in two exposure times. Seven test microorganisms were prepared and inoculated on the surface of seven types of PPE (respirator mask, face shield, shoe, glove, cap, safety glasses and lab coat). The tests were performed on previously contaminated PPE using a manikin with a motion device for exposure to the chamber with biocidal agent (sodium hypochlorite) for 10 and 30s. In 96.93% of the experimental conditions analyzed, the percentage reduction was >99% (the number of viable cells found on the surface ranged from 4.3x10⁶ to <10 CFU/mL). The samples of E. faecalis collected from the glove showed the lowest percentages reduction, with 86.000 and 86.500% for exposure times of 10 and 30 s, respectively. The log₁₀ reduction values varied between 0.85 log₁₀ (E. faecalis at 30 s in glove surface) and 9.69 log₁₀ (E. coli at 10 and 30 s in lab coat surface). In general, E. coli, S. aureus, C. freundii, P. mirabilis, C. albicans and C. parapsilosis showed susceptibility to the biocidal agent under the tested conditions, with >99% reduction after 10 and 30s, while E. faecalis and P. aeruginosa showed a lower susceptibility. The 30s exposure time was more effective for the inactivation of the tested microorganisms. The results show that the spray disinfection technology has the potential for instant decontamination of PPE, which can contribute to an additional barrier for infection control of healthcare workers in the hospital environment.