Current guidelines recommend reflex testing for hepatitis D virus (HDV) coinfection in hepatitis B surface antigen (HBsAg)-positive patients over risk-factor based screening. We aimed to evaluate the feasibility and diagnostic yield of reflex anti-HDV testing at a Central European tertiary care center. We retrospectively included 560 consecutive patients who had a recorded (first) positive HBsAg test result at the Vienna General Hospital between 2018 and 2022. While reflex anti-HDV testing had been implemented in our hepatitis outpatient clinic (n = 153, ‘reflex testing cohort’), HDV screening needed to be manually ordered in the remaining patients (n = 407, ‘standard testing cohort’). Overall, 98.0% and 65.1% of patients in the reflex and standard testing cohort were screened for anti-HDV, respectively, and the overall seroprevalence of anti-HDV among screened patients was 6.7% (n = 28, reflex testing cohort: 9.3%, standard testing cohort: 5.3%). Risk factors for HDV were present in 49.1% of all included and in 89.3% of anti-HDV positive patients, respectively. Anti-HDV positive patients showed higher ALT (54 [33–83] vs. 29 [19–49] U/L; p = 0.005) and a higher proportion of low-to-undetectable HBV-DNA (61.5% vs. 33.2%; p < 0.001), as compared to anti-HDV negative patients. HDV-RNA PCR was ordered in n = 21/28 (75.0%) of anti-HDV positive patients, and 76.2% had detectable HDV-RNA. Among viremic patients, 75% and 37.5% had significant fibrosis (≥ F2) or cirrhosis (F4), respectively. The prevalence of anti-HDV among HBsAg-positive patients is considerable in a large hospital located in Central Europe. Double reflex testing, i.e., anti-HDV being triggered by the presence of HBsAg and HDV-PCR bring triggered by the presence of anti-HDV, seems warranted to increase the diagnostic yield.