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Over one hundred bacterial species have been determined to comprise the human microbiota in a healthy individual. Bacteria including <i>Escherichia coli</i>, <i>Bacillus cereus</i>, <i>Clostridioides difficile</i>, and <i>Vibrio parahaemolyticus</i> are found inside of the human body and <i>B. cereus</i> and <i>E. coli</i> are also found on the skin. These bacteria can act as human pathogens upon ingestion of contaminated food or water, if they enter an open wound, or antibiotics, and environment or stress can alter the microbiome. In this study, we present new polymerase chain reaction (PCR) high-resolution melt (HRM) assays to detect and identify the above microorganisms. Amplified DNA from <i>C. difficile</i>, <i>E. coli, B. cereus</i>, and <i>V. parahaemolyticus</i> melted at 80.37 ± 0.45 °C, 82.15 ± 0.37 °C, 84.43 ± 0.50 °C, and 86.74 ± 0.65 °C, respectively. A triplex PCR assay was developed to simultaneously detect and identify <i>E. coli</i>, <i>B. cereus</i>, and <i>V. parahaemolyticus</i>, and cultured microorganisms were successfully amplified, detected, and identified. The assays demonstrated sensitivity, specificity, reproducibility, and robustness in testing.