The use of handheld Raman spectroscopy has increased in popularity based on its ability to provide portable and rapid analysis of endogenous compounds and diagnostic biomarkers in alternative biological matrices, such as fingernails. The application of fingernails as a diagnostic matrix allows for non-invasive, non-intrusive sampling, which can be carried out in the comfort of the patient’s home. This study aimed to identify diagnostic biomarkers in fingernails related to cardiovascular diseases (CVDs) and diabetes mellitus (DM) using Raman spectroscopy and machine learning algorithms (MLAs). The findings showed that Raman spectroscopy successfully identified the presence of disease specific biomarkers in CVD and diabetic fingernails. Furthermore, when used in combination with MLAs, Raman spectroscopy was able to differentiate between healthy, CVD and diabetic fingernails. Further investigation will look at applying additional MLAs for determining the prognosis of disease.