The objective of this study was to evaluate the performance of the Copan Colibrí™ against the manual preparation of the MALDI targets. We analyzed 416 (31 different species) non-duplicate strains covering the most important species identified in clinical routine. We also assessed the intra-strain repeatability between the comparable methods. We then analyzed the performance of this new method after implementation in routine on 12,253 aerobic bacterial isolates and yeasts, encompassing a total of 42 different species. Among the 416 strains analyzed, 6.3% (26/416) and 10.8% (45/416) had a score value < 2 when processed by the Colibri™ and manual method, respectively. Only 5.9% (9/152) of the Gram positive rods and cocci had a score values < 2 by the Colibri™ versus 20.4% (31/152) by the manual method. We confirmed that this relative superiority observed for the Colibri™ was due primarily in the use of the formic acid protocol. For the Gram-negative bacteria, the results of both methods were comparable; 6.6% (17/256) and 4.7% (12/256) had a score value < 2 by the Colibri™ and the manual method, respectively. After implementation in routine, the results according to the Biotyper score cut-off values were distributed as follows: < 1.70: 2.5% (304/12,253), 1.70–1.79: 1.9% (227/12,253), 1.80–1.89: 3.1% (377/12,253), 1.90–1.99: 6.7% (825/12,253), and ≥ 2: 85.9% (10,520/12,253). The Colibrí™ coupled to MALDI-TOF/MS revealed good performances and higher intra-strain repeatability as compared to the manual preparation of the MALDI targets.