Ensuring hygiene in dairy processing facilities is essential for maintaining food safety and preventing microbial contamination. Traditional microbiological methods, such as plate count techniques, require long incubation times and are unsuitable for rapid hygiene assessments. This study evaluates the application of ATP bioluminescence as a real-time monitoring tool and compares its effectiveness with classical microbiological techniques in a dairy processing plant.Sampling was conducted over three months in six critical locations, including the raw milk reception area, pasteurization equipment, filling machine nozzles, conveyor belts, storage tanks, and operator’s gloves. The microbial contamination was assessed using both ATP bioluminescence measurements (expressed in relative light units, RLU) and total viable count (TVC) measurements (expressed in CFU/cm²). The correlation between ATP readings and microbial loads before and after cleaning procedures was analyzed to determine the reliability of ATP testing in hygiene control.Results demonstrated a significant reduction in contamination after cleaning, with ATP RLU values decreasing by 75–90% and CFU levels showing similar declines. The Pearson correlation coefficient (R) between ATP values and TVC counts increased from 0.001–0.600 before cleaning to 0.720–0.990 after cleaning, confirming a strong relationship between the two methods.The study highlights ATP bioluminescence as a rapid and efficient tool for hygiene monitoring in dairy plants. However, ATP testing does not differentiate between viable and non-viable microorganisms, necessitating additional microbiological confirmation for precise contamination assessment. Despite this limitation, its ability to provide immediate feedback allows for timely corrective actions, making it a valuable component of dairy hygiene management programs. Integrating ATP bioluminescence with traditional microbiological techniques can enhance food safety, optimize sanitation procedures, and ensure compliance with industry regulations.