Background The use of negative and positive controls, such as bacterial mock community, is a crucial part of a well-established workflow for 16S rDNA sequencing. Several mock community standards (i.e., mixes with known composition of bacterial species) are available for purchase. To prevent interference with the sample, the selected mock community should ideally consist of species not occurring in the target sample. We aimed to set up a method for analysis of bacteriome of three human low-abundance matrices that takes advantage of spiking of these samples with non-human mock community in amounts not overlaying the sample bacteriome but, and at the same time, yielding sufficient sequence coverage. Methods Samples of urine, blood and bronchoalveolar lavage (BAL) from 3 patients, 3 bacterial cultures of Klebsiella pneumoniae (104 CFU/mL) serving as positive controls, and 3 negative controls (DNA free water) were isolated by QIAamp DNA Blood Mini Kit in triplicates. Parallelly, the mock community ZymoBIOMICS Spike-in Control I (High Microbial Load, MOCK), consisting of bacterial species Allobacillus and Imtechella (1:1), was isolated. All samples and controls were subsequently spiked with 1 µl of 500×, 1,000× or 2,000× diluted DNA from isolated mock (what more 3 negative controls without spiking were also used) and amplified. Two PCRs were performed – one with 30 and the other with 33 cycles. The final library was sequenced on the MiSeq instrument using MiSeq Reagent Kit v3 (600-cycle). Results The sequencing analysis was successful with both 30 and 33 cycles used for PCR. The results showed the contamination of reagents with Flavobacterium (~1,000 reads per sample) and Massilia (~250 reads per sample) species. These bacteria were, therefore, excluded from subsequent data analysis. In urine, blood, and BAL samples with 2,000× diluted mock, the average relative abundance of Allobacillus and Imtechella was 5 % (on average 5 % combined), while with the 1,000× and 500× diluted mock, the relative abundances of these mock community bacteria were 30 % and 60 % (on average), respectively. Conclusions 16S rDNA sequencing of urine, blood and BAL samples spiked with mock was successfully performed; however, we detected the presence of certain bacterial DNA in used reagents. For the studied matrices, we recommend: i) to monitor the possibility of sample contamination from reagents; ii) to use the mock dilution for these lowabundance matrices of 2,000×, which results in approx. 5 % relative abundance of the mock community in the sample. This mock dilution is sufficient to serve as an internal standard while not overlaying the bacteria, which are abundant more than 5 % in this type of samples.