Contamination of environmental surfaces by nosocomial pathogens like (), (), and spp. poses significant health risks worldwide. However, gold-standard detection methods are too time-consuming and labor-intensive. This study aimed to optimize loop-mediated isothermal amplification (LAMP) as a rapid, innovative, and cost-effective approach, comparing its effectiveness with the gold-standard cultural method. Sterile surfaces (24 cm) were contaminated in duplicate with different concentrations of , , and reference stains. For each pair of contaminated surfaces, one was analyzed using the agar contact plate method (UNI EN 17141:2021), while the other was analyzed using LAMP, following three different pre-incubation times (three, six, and nine hours). The sensitivity and accuracy of LAMP for improved with longer incubation times, reaching a value of 1.00 at nine hours, while the specificity and positive predictive value (PPV) remained at 1.00 regardless of the incubation time. For , LAMP achieved a sensitivity, specificity, accuracy, PPV, and negative predictive value (NPV) of 1.00 across all incubation times. Finally, for , sensitivity increased from 0.57 at three hours to 1.00 at six and nine hours, with a high specificity, accuracy, PPV, and NPV from six hours onwards. These findings showed that LAMP can be used as a rapid and reliable alternative to gold-standard methods for detecting pathogens on surfaces. The high sensitivity and specificity achieved, especially at six and nine hours of pre-incubation, suggested its use for real-time monitoring in healthcare settings. Further research in real-world environments is needed to confirm these findings.