Efficient methods for constructing 16S tag amplicon libraries for pyrosequencing are needed for the rapid and thorough screening of infectious bacterial diversity from host tissue samples. Here we have developed a double-nested PCR methodology that generates 16S tag amplicon libraries from very small amounts of bacteria/host samples. This methodology was tested for 133 kidney samples from the lake whitefish Coregonus clupeaformis (Salmonidae) sampled in five different lake populations. The double-nested PCR efficiency was compared with two other PCR strategies: single primer pair amplification and simple nested PCR. The double-nested PCR was the only amplification strategy to provide highly specific amplification of bacterial DNA. The resulting 16S amplicon libraries were synthesized and pyrosequenced using 454 FLX technology to analyse the variation of pathogenic bacteria abundance. The proportion of the community sequenced was very high (Good’s coverage estimator; mean = 95.4%). Furthermore, there were no significant differences of sequence coverage among samples. Finally, the occurrence of chimeric amplicons was very low. Therefore, the double-nested PCR approach provides a rapid, informative and cost-effective method for screening fish immunobiomes and most likely applicable to other low-density microbiomes as well.