Contaminated surfaces increase the risk of hospital infections. Traditional hospital cleanliness monitoring has become insufficient. ATP bioluminescence is a developed monitoring tool with limited clinical data in healthcare settings. Therefore, the current work aims to study the impact of the ATP monitoring tool on wound infection rates and fecal colonization among burn patients. The study was designed over two phases. Phase I involved conventional cleaning monitoring by visual inspection, while phase II involved the ATP bioluminescence tool. In both phases, clinical and environmental swabs were collected for microbial culture and identification. Gram-negative bacteria were screened for carbapenem resistance. Among the five selected cases, MALDI-TOF and Vitek2 were utilized to test for phenotypic relatedness between common isolates from different clinical and environmental sources. The wound infection rate was significantly reduced from 23% in phase I to 8% in phase II (p-value <0.005). Fecal colonization by CR bacteria demonstrated 7% and 14% in phase I and phase II, respectively. Environmental culture demonstrated significantly decreased microbial isolation rates from 37% (phase I) to 10% (phase II) (p-value<0.001) with a non-significant decrease in CR bacteria. Total pass and failed cleaning rates for ATP bioluminescence were 70.9% and 6.08%, respectively. Common isolates in 3 cases exhibited a similarity of >65% by MALDI-TOF and the identical resistance phenotypes by Vitek2. The ATP bioluminescence cleaning verification system has been proven a rapid and objective tool that positively impacts microbial isolation rates from clinical and environmental samples.