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Background Antitrypanosomal treatment with Benznidazole (BZ) or Nifurtimox may be recommended for patients with chronic Chagas disease (CD) to reduce the onset or progression of symptoms. However, such treatment has limited efficacy and high level of toxic effects. In addition, the current cure biomarker (serology conversion) precludes any treatment assessment unless a prolonged follow-up is arranged. PCR is thus the most useful, alternative surrogate marker for evaluating responses to treatment. The aim of this study is to describe the usefulness of real-time PCR in monitoring BZ treatment within a large cohort of chronic CD cases in Barcelona. Methodology/Principal findings A total of 370 chronic CD patients were monitored with real-time PCR post-BZ treatment. The median follow-up was 4 years (IQR 2.2–5.3y), with a median of 3 clinical visits (IQR 2–4). Only 8 patients (2.2%) presented with at least one incident of positive real-time PCR after treatment and were therefore considered as treatment failure. Four of those failure patients had completed full course treatment, whereas the remaining cases had defaulted with a statistical difference between both groups (p = 0.02). Half of the failure patients had undergone less than 4 years of follow-up monitoring all presented with parasitemia before treatment. Conclusions/Significance BZ treatment failure was highly infrequent in our cohort. BZ discontinuation was a risk factor for positive real-time PCR results during clinical follow-up. Regular testing with real-time PCR during follow-up allows for early detection of treatment failure in patients with chronic CD., Author summary Chagas disease has become a public health concern for health services in Spain, provided that the destination is the primary choice for Latin American migrants in Europe. Chronic phase is the most frequent clinical form of the disease outside of endemic areas. Most of these patients usually undergo treatment with Benznidazole, primarily childbearing women. Monitoring response to antiparasitic treatment without the presence of an early cure biomarker poses as one of the greatest challenges in managing this disease. In this study, almost 400 chronic Chagas disease patients treated with Benznidazole in two referral centers in Barcelona were included. Real-time PCR and serology were performed in all clinical visits after treatment; real-time PCR has been used as a surrogate biomarker to assess response to treatment. The failure rate of Benznidazole was very low in our cohort, with treatment discontinuation being a risk factor for such failure. In summary, our data supports that regular testing by real- time PCR for monitoring treatment response in chronic Chagas disease patients could be a useful surrogate marker for failure in such individuals.