High biosecurity standards are essential in poultry farms, where cleaning and disinfection are key to maintaining hygiene and animal health. Evaluating these procedures requires rapid and reliable detection methods. Plate cultivation remains the gold standard but is time-consuming and limited to culturable microorganisms, while luminometers mainly detect organic contamination. This study evaluated the potential of a mobile flow cytometer for hygiene monitoring on a broiler farm. The device (CytoQuant, Romer Labs) uses impedance and alternating current at multiple frequencies to detect and quantify intact cells and particles without optical systems. This study was conducted on a commercial broiler farm. Samples were collected during sanitation from three sites (drinker, concrete wall, and metal wall) by swabbing 100 cm² surfaces. Measurements followed the manufacturer’s protocol. Plate cultivation determined total microbial counts (TMCs) at 22 °C (plate count agar, PCA) and 36 °C (blood agar, BA). Correlations were assessed using the Spearman correlation coefficient (rSp). Significant correlations were found for the metal wall (BA: rSp = 0.8117, p < 0.01; PCA: rSp = 0.8333, p < 0.01) and the concrete wall (BA: rSp = 0.8000, p < 0.01; PCA: rSp = 0.7667, p < 0.05), but not for drinkers. The cytometer detected higher microbial counts than cultivation, by 1.13–2.26 log CFU on BA and 0.76–1.02 log CFU on PCA (p < 0.05, Wilcoxon test). This difference reflects enumeration of all intact cells, unlike plate methods, which detect only culturable ones. Environmental hygiene is crucial for broiler health, welfare, and productivity. The mobile flow cytometer demonstrated strong potential for assessing sanitation efficiency and may complement plate methods. Unlike luminometry, the results are unaffected by disinfectant residues. Flow cytometry provides a rapid, practical tool for hygiene monitoring, enhancing biosecurity and supporting One Health strategies by reducing microbial loads and optimizing disinfectant use.