Background A proportion of patients with Lyme borreliosis (LB) report long-term persisting signs and symptoms, even after recommended antibiotic treatment, which is termed post-treatment Lyme disease syndrome (PTLDS). Consensus on guidance regarding diagnosis and treatment is currently lacking. Consequently, patients suffer and are left searching for answers, negatively impacting their quality of life and healthcare expenditure. Yet, health economic data on PTLDS remain scarce. The aim of this article is therefore to assess the cost-of-illness related to PTLDS, including the patient perspective. Methods PTLDS patients (N = 187) with confirmed diagnosis of LB were recruited by a patient organization. Patients completed a self-reported questionnaire on LB-related healthcare utilization, absence from work and unemployment. Unit costs (reference year 2018) were obtained from national databases and published literature. Mean costs and uncertainty intervals were calculated via bootstrapping. Data were extrapolated to the Belgian population. Generalized linear models were used to determine associated covariates with total direct costs and out-of-pocket expenditures. Results Mean annual direct costs amounted to euro4618 (95% CI euro4070-5152), of which 49.5% were out-of-pocket expenditures. Mean annual indirect costs amounted to euro36 081 (euro31 312-40 923). Direct and indirect costs at the population level were estimated at euro19.4 and 151.5 million, respectively. A sickness or disability benefit as source of income was associated with higher direct and out-of-pocket costs. Conclusions The economic burden associated with PTLDS on patients and society is substantial, with patients consuming large amounts of non-reimbursed healthcare resources. Guidance on adequate diagnosis and treatment of PTLDS is needed.