Implementation of care bundles for prevention of ventilator-associated pneumonia (VAP) and its impact on patient outcomes requires validation with long-term follow-up. A collaborative multi-centre cohort study was conducted in five Spanish adult intensive-care units. A care bundle approach based on five measures was implemented after a 3-month baseline period, and compliance, VAP rates, intensive-care unit length of stay (ICU LOS) and duration of mechanical ventilation were prospectively recorded for 16 months. There were 149 patients in the baseline period and 885 after the intervention. Compliance with all measures after intervention was <30% (264/885). In spite of this, VAP incidence decreased from 15.5% (23/149) to 11.7% (104/885), after the intervention (p <0.05). This reduction was significantly associated with hand hygiene (OR = 0.35), intra-cuff pressure control (OR = 0.21), oral hygiene (OR = 0.23) and sedation control (OR = 0.51). Use of the care bundle was associated with an incidence risk ratio of VAP of 0.78 (95% CI 0.15–0.99). We documented a reduction of median ICU LOS (from 10 to 6 days) and duration of mechanical ventilation (from 8 to 4 days) for patients with full bundle compliance (intervention period). Efforts on VAP prevention and outcome improvement should focus on achieving higher compliance in hand and oral hygiene, sedation protocols and intracuff pressure control.