Disinfectants are used intensively to control and prevent healthcare-associated infections. With continuous use and exposure to disinfectants, bacteria may develop reduced susceptibility. The study aimed to check the hygiene of devices in the physiotherapy department. For isolated bacterial strains, we aimed to determine the minimum inhibitory concentration of five different disinfectant wipe products currently in use. Microbiological environmental sampling in four various institutions in four different cities from two counties was performed, followed by CFU calculation and identification using matrix-assisted laser desorption and ionization with time-of-flight analyzer mass spectrometry (MALDI-TOF). The sampling was performed on three different occasions: before patient use, after patient use, and after disinfection. The susceptibility of isolates to three different alcohol-based and three different quaternary ammonium compounds (QAC) disinfectant wipes was examined by determining the minimal inhibitory concentrations (MIC). We identified 27 different bacterial species from 11 different genera. Gram-positive bacteria predominated. The most abundant genera were , , and . The average MIC values of alcohol-based disinfectants range between 66.61 and 148.82 g/L, and those of QAC-based disinfectants range between 2.4 and 3.5 mg/L. Distinctive strains with four-fold increases in MIC values, compared to average values, were identified. The widespread use of disinfectants can induce a reduction in the susceptibility of bacteria against disinfectants and affect the increase in the proportion of antibiotic-resistant bacteria. Therefore, it is urgent to define clear criteria for defining a microorganism as resistant to disinfectants by setting epidemiological cut-off (ECOFF) values and standardizing protocols for testing the resistance of microorganisms against disinfectants.