Abstract Background Antibiotic-impregnated external ventricular drains (AI-EVDs) have a debated efficacy in clinical studies. Objectives Our aim was to assess the durability of antimicrobial activity of AI-EVDs used in clinical settings. Methods From April 2017 to January 2018, all consecutive AI-EVDs (Bactiseal™) inserted in adult patients were prospectively included. After removal, each AI-EVD was cultured and assessed for antimicrobial activity on both internal and external sides of AI-EVDs. Catheters were each challenged with a single Staphylococcus strain [MSSA, MRSA or methicillin-resistant Staphylococcus epidermidis (MRSE)]. MS was used to measure residual concentrations of rifampicin and clindamycin. Results Sixty-five AI-EVDs were included (56 patients). Among these, 21 were challenged with MSSA, 23 with MRSA and 21 with MRSE. Five ventriculostomy-related colonizations (9%) and two ventriculostomy-related infections (4%) occurred. Staphylococcus was the main bacterium responsible for colonization (4/5). AI-EVD inhibition decreased significantly against MRSA and MRSE according to duration of catheterization (for external and internal sides, P < 0.02) and overall volume of CSF drained (P < 0.005 for both sides against MRSE, P < 0.005 for external side against MRSA), but not against MSSA. Clindamycin concentration was not correlated with duration of catheterization or CSF volume drained, but <20% of initial concentration was recovered even after 5 days of AI-EVD dwelling. Conversely, rifampicin concentration showed a rapid and significant decline correlated to duration and CSF volume (P < 0.001 and P = 0.03, respectively). Conclusions Antimicrobial activity of AI-EVDs dropped quickly in vivo. Antimicrobial impregnation did not prevent AI-EVD colonization by susceptible strains in 9% of the cases.