Allostatic load is a model that is used to quantify the physiological damage from exposure to stressors. Stressful life events are chronic stressors that can lead to an elevated allostatic load through the physiological and behavioral stress responses. However, there is limited empirical studies that has tested the proposed behavioural pathway. Our study addresses this gap by examining the mediating role of combined modifiable lifestyle behaviors in the 12-years longitudinal association between stressful life events and allostatic load among participants from the Australian Diabetes, Obesity and Lifestyle (AusDiab) Study cohort. A latent profile analysis was performed to identify latent subgroups with distinct behavioral clusters based on five modifiable lifestyle behaviors (smoking, sedentary behavior, physical activity, alcohol consumption, and diet quality). We then used a sequential mediation model design with path analysis to test the mediating effect of these latent subgroups in the associations between stressful life events and three measures of allostatic load. Indirect effects were estimated using the product of coefficient approach and the statistical significance was determined by the 95% bias-corrected bootstrap confidence intervals with 1000 replications. We identified three latent subgroups: “least healthy lifestyle” (12%; n = 396), “moderately healthy lifestyle” (78.7%; n = 2599), and “most healthy lifestyle” (9.2%; n = 306). Exposure to stressful life events was not associated with the allocation of participants in latent subgroups. Compared to the “moderately healthy lifestyle” subgroups, we found that the “least healthy lifestyle” behavioral cluster was not associated with allostatic load. However, there was a significant inverse association between the “most healthy lifestyle” behavioral cluster and allostatic load. Overall, we did not find significant indirect effects between stressful life events and three measures of allostatic load via the “least healthy lifestyle” and the “most healthy lifestyle” groups. In summary, the combinations of modifiable lifestyle behaviors did not explain the association between stressful life events and allostatic load. More longitudinal studies are needed to replicate our study to confirm this finding