The effect of tenofovir (TDF) alone or in combination with interferon on hepatitis D virus (HDV) replication is poorly characterized in patients infected with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and HDV. We analyzed triinfected patients undergoing treatment with either TDF alone (n=13) or including interferon (IFN) at some point during TDF therapy (TDF+IFN, n=4). Linear mixed-effect models were used to estimate the mean change from baseline of HDV-RNA and hepatitis surface antigen (HBsAg) levels during treatment. Patients were followed for a median 31.6 (25-75%-tile: 15.0-47.4) months. In the TDF+IFN group, three initiated IFN-based therapy after a median of 21.7 months (range=10.5-24.9) of lamivudine (LAM)+TDF, while the remaining patient had 46.8 months of prior LAM exposure. Significant decreases in HDV-RNA were observed in both groups [TDF alone: -0.380 log10 copies/ml per year (95% CI: -0.557, -0.202) vs. TDF+IFN: -1.325 log10 copies/ml per year (95% CI: -1.931, -0.720)], while the HDV-RNA decline overall was significantly faster in patients with TDF+IFN (p=0.002). Accordingly, two patients achieved HDV-RNA below the limit of quantification (LOQ: <1,000 copies/ml) and one near LOQ (1450 copies/ml), all concomitantly treated with interferon. There were no significant changes in HBsAg levels for either group [TDF alone: -0.008 log10 IU/ml per month (95% CI: -0.019, 0.004), TDF+IFN:-0.011 log10 IU/ml per month (95% CI: -0.037, 0.015)] and no significant difference in slope between treatment groups (p=0.8). Interferon therapy might be more effective after extended previous anti-HBV antiviral exposure among triinfected patients; however, the long-term implications of these findings remain unknown.