Background: Direct-acting antiviral agents (DAAs) cure patients with hepatitis C virus (HCV) infection. Concerns have arisen the occurrence of significant bradyarrhythmias during treatment with DAAs. The aim of this study was to assess the impact of a DAA combination for the treatment of HCV infection on heart rate, rhythm, and heart rate variability (HRV) using 24-h ECG monitoring. Results: A prospective randomized study of 50 treatment-naïve patients with HCV infection treated with a combination of sofosbuvir 400 mg daily and daclatasvir 60 mg daily for 12 weeks. Surface ECG and 24-h ECG monitoring were performed at baseline and after completion of therapy to assess PR interval, corrected QT interval (QTc), minimum heart rate (HR), maximum HR, average HR, HRV time-domain and frequency-domain measures, significant pauses, tachycardias, bradycardias, premature atrial contractions (PACs), and premature ventricular contraction (PVCs). No differences were detected in all examined parameters between baseline and after completion of treatment. PR interval was 154 ± 25.95 vs 151.4 ± 23.82 ms, respectively (p = 0.124). QTc interval was 397.34 ± 29.38 vs 395.04 ± 30.23 ms, respectively (p = 0.403). No differences were detected for minimum HR, maximum HR, average HR, HRV time-domain and frequency-domain measures, the occurrence of significant pauses, sinus tachycardia episodes, sinus bradycardia episodes, PACs, and PVCs. No episodes of bradyarrhythmias, syncope, and atrial fibrillation, supraventricular, or ventricular tachycardias were reported or detected. Conclusion: In non-cardiac patients receiving no cardioactive medications, the combination of sofosbuvir and daclatasvir for the treatment of HCV infection has no effect on HR, rhythm, conductivity, or HRV. No symptomatic bradycardias, tachycardias, or syncope were reported or detected using 24-h ECG monitoring.