Per- and polyfluoroalkyl substances (PFASs) are synthetic chemicals widely applied in industrial processes and consumer products due to their thermal stability and resistance to degradation. However, these same properties make PFASs environmentally persistent and a potential threat to human health. Although international concern about PFAS exposure has grown, limited research has explored their effects in African populations. This study investigated the serum levels of three PFASs—perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS), and perfluorobutane sulfonate (PFBS)—and their association with diabetes mellitus (DM) in a mixed-ancestry population from Bellville South, Cape Town, South Africa. Serum samples (n = 179) were analyzed using liquid chromatography–tandem mass spectrometry (LC–MS-8030), and statistical analyses were performed with STATISTICA software. All three PFASs were detected in every sample, with PFOA showing the highest mean concentration (9.43 ± 13.16 ng/mL). Concentrations were generally higher in females than males, with PFOA levels significantly elevated among women (p = 0.0116). Despite these exposures, no statistically significant associations were observed between PFAS concentrations and glycaemic status, obesity, or other metabolic predictors (p > 0.05). Notably, PFOS demonstrated a modest positive correlation with HbA1c in females, suggesting potential gender-specific interactions. While our findings did not establish a clear link between PFAS exposure and DM, they highlight measurable human exposure within this community and emphasize the need for further research. Given the widespread detection of PFASs in environmental matrices and medicinal plants in South Africa, larger-scale longitudinal studies are warranted to clarify exposure pathways and potential health implications.