BACKGROUND: The aim of this study is to apply random forest (RF), one of the machine learning (ML) algorithms, to a dataset consisting of patients with a presumed diagnosis of acute appendicitis (AAp) and to reveal the most important factors associated with the diagnosis of AAp based on the variable importance. METHODS: An open-access dataset comparing two patient groups with (n=40) and without (n=44) AAp to predict biomarkers for AAp was used for this case−control study. RF was used for modeling the data set. The data were divided into two training and test dataset (80:20). Accuracy, balanced accuracy (BC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) performance metrics were appraised for model performance. RESULTS: Accuracy, BC, sensitivity, specificity, PPV, NPV, and F1 scores pertaining to the RF model were 93.8%, 93.8%, 87.5%, 100%, 100%, 88.9%, and 93.3%, respectively. Following the variable importance values regarding the model, the variables most associated with the diagnosis and prediction of AAp were fecal calprotectin (100 %), radiological imaging (89.9%), white blood test (51.8%), C-reactive protein (47.1%), from symptoms onset to the hospital visit (19.3%), patients age (18.4%), alanine aminotransferase levels >40 (<1%), fever (<1%), and nausea/vomiting (<1%), respectively. CONCLUSION: A prediction model was developed for AAp with the ML method in this study. Thanks to this model, biomarkers that predict AAp with high accuracy were determined. Thus, the decision-making process of clinicians for diagnosing AAp will be facilitated, and the risks of perforation and unnecessary operations will be minimized thanks to the timely diagnosis with high accuracy.