Rapid identification of the etiological agent in bacterial infection is necessary for correct diagnosis and appropriate therapy. In general, identification of pure cultures of bacteria using conventional phenotyping techniques requires 4-24 hours. Recently available new molecular technologies offer the potential of same day species identification once pure culture is available. Our aim was to evaluate the performance of rDNA V1 hypervariable region pyrosequencing, and the whole cell MALDI-TOF MS protein profiling in routine species identification. During the period from June 2012 to June 2014, 1.140 pure culture isolates were recovered from 402 samples from 126 patients suffering cystic fibrosis, chronic obstructive pulmonary disease or bronchiectasis. All the isolates were subjected to species identification by both techniques. Unfortunately, pyrosequencing was able to reach the species level in 43.2% of isolates only, whereas MALDI-TOF was clearly superior with 96.8% respectively. The overall sensitivity values also clearly underlined the superiority of MALDI-TOF MS with 96.8% compared to 85.1% achieved by pyrosequencing. Generally, MALDI-TOF MS turned out to be the best suitable technique in routine bacterial identification, whereas pyrosequencing could be recommended as the method of choice particularly in situations where MALDI-TOF MS fails to identify rare species.