It is essential to refrain from unnecessary isolation measures indicated for patients identified with multidrug-resistant Gram-negative bacteria (MDR-GNB). To evaluate whether a pro-active follow-up strategy to discontinue isolation measures of patients identified with MDR-GNB (without carbapenemase production) resulted in reduced isolation days during hospitalization, compared to passive follow-up. A comparison was made between active and passive follow-up strategies over a two-year period after first MDR-GNB identification. Patients could be declared negative after two consecutive negative screening cultures. Active follow-up patients received a questionnaire for screening cultures within six months of MDR-GNB identification. Of the 2208 patients included, 1424 patients (64.5%) underwent passive follow-up and 784 patients (35.5%) underwent active follow-up. A significantly higher proportion of active follow-up patients who had sufficient (at least two) screening cultures were declared MDR-GNB negative compared to those with passive follow-up; 66.9% vs 20.6% (P < 0.001) for adult patients and 76.0% vs 17.1% (P < 0.001) for paediatric patients. A comparison between active follow-up patients with sufficient versus those with active follow-up but insufficient cultures revealed a reduction of isolation days for paediatric patients (median 10.6 vs 1.6 days; P= 0.031). Though this difference was not statistically significant for adults (median 5.3 vs 4.2 isolation days), there was a valuable decrease in the number of isolation days for both adult and paediatric patients under active follow-up with sufficient (≥2) cultures, indicating clinical relevance. We recommend an active follow-up strategy for patients identified with an MDR-GNB, to prevent further unneeded infection prevention measures.