The co-occurrence of chronic illnesses and substance use presents complex challenges for health care systems. Understanding the interplay between these factors, compounded by the context of the COVID-19 pandemic, is essential for effective intervention strategies. This study aims to investigate the relationships among chronic illness, substance use, and COVID-19 infection in adults aged 50 years and older. Participants were 1196 adults aged 50 years and older. Descriptive statistics were used to describe demographic information. Logistic regressions and multiple regression analyses were used to determine associations between chronic illnesses, substance use, and COVID-19 infection. Mediation analysis was used to determine the effect of chronic illness mediators in the association between COVID-19 concerns and substance use. The mean age was 68 (SD 10.3) years, with 58.6% (701/1196) being women. Adjusted analysis revealed that age and sex (women) significantly predicted a lower level of substance use (P<.05). However, marital status (separated or widowed) and chronic illness significantly predicted a higher level of substance use (P<.05). Furthermore, having dementia, arthritis, and high cholesterol significantly predicted a higher level of concern about the COVID-19 pandemic (P<.05). Logistic regression analysis indicated that individuals with hypertension (odds ratio [OR] 1.91, 95% CI 1.37-2.66; P<.001), lung disease (OR 2.42, 95% CI 1.23-4.75; P=.01), heart condition (OR 1.99, 95% CI 1.28-3.10; P=.002), stroke (OR 2.35, 95% CI 1.07-5.16; P=.03), and arthritis (OR 1.72, 95% CI 1.25-2.37; P=.001) were more likely to have their work affected by the COVID-19 pandemic. The mediation analysis showed a significant effect of COVID-19 concern on substance use through the mediation of chronic illness, with a 95% CI of -0.02 to -0.01 and an indirect effect of -0.01. Our study reveals complex associations among chronic illnesses, substance use, and COVID-19 infection among adults aged 50 years and older. It underscores the impact of demographics and specific chronic conditions on substance use behaviors and COVID-19 concerns. In addition, certain chronic illnesses were linked to heightened vulnerability in employment status during the pandemic. These findings emphasize the need for targeted interventions addressing physical health and substance use in this population during the COVID-19 pandemic.