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Ensuring dairy product safety demands rapid and precise Staphylococcus aureus (S. aureus) detection. Biosensors show promise, but their performance is often demonstrated in model samples using non-native pathogens and has never been studied towards S. aureus detection in naturally contaminated samples. This study addresses the gap by directly comparing results taken with a novel piezoelectric biosensor, capable of one-step detection, with four conventional cultivation-based methods. Our findings reveal that this biosensor, based on an antifouling nanolayer-coated biochip, exhibits exceptional resistance to biofouling from unprocessed dairy products and is further capable of specific S. aureus detection. Notably, it performed comparably to Petrifilm and Baird-Parker methods but delivered results in only 30 min, bringing a substantial reduction from the 24 h required by cultivation-based techniques. Our study also highlights differences in the performance of cultivation methods when analyzing artificially spiked versus naturally contaminated foods. These findings underline the potential of antifouling biosensors as efficient reliable tools for rapid, cost-effective, point-of-care testing, enhancing fresh dairy product safety and S. aureus detection.