Abstract Background The burden of human immunodeficiency virus (HIV) infection in people who use drugs (PWUD) is significant. We aimed to screen HIV infection among PWUD and describe their retention in HIV care. Besides, we also screen for hepatitis C virus (HCV) infection among HIV-seropositive PWUD and describe their linkage to care. Methods We conducted a prospective study in 529 PWUD who visited the “Cañada Real Galiana” (Madrid, Spain). The study period was from June 1, 2017, to May 31, 2018. HIV diagnosis was performed with a rapid antibody screening test at the point-of-care (POC) and HCV diagnosis with immunoassay and PCR tests on dried blood spot (DBS) in a central laboratory. Positive PWUD were referred to the hospital. We used the Chi-square or Fisher’s exact tests, as appropriate, to compare rates between groups. Results Thirty-five (6.6%) participants were positive HIV antibodies, but 34 reported previous HIV diagnoses, and 27 (76%) had prior antiretroviral therapy. Among patients with a positive HIV antibody test, we also found a higher prevalence of homeless (P < 0.001) and injection drug use (PWID) (P < 0.001), and more decades of drug use (P = 0.002). All participants received HIV test results at the POC. Of the 35 HIV positives, 28 (80%) were retained in HIV medical care at the end of the HIV screening study (2018), and only 22 (62.9%) at the end of 2020. Moreover, 12/35 (34.3%) were positive for the HCV RNA test. Of the latter, 10/12 (83.3%) were contacted to deliver the HCV results test (delivery time of 19 days), 5/12 (41.7%) had an appointment and were attended at the hospital and started HCV therapy, and only 4/12 (33.3%) cleared HCV. Conclusions We found almost no new HIV-infected PWUD, but their cascade of HIV care was low and remains a challenge in this population at risk. The high frequency of active hepatitis C in HIV-infected PWUD reflects the need for HCV screening and reinforcing the link to care.