In this study three DNA extraction procedures, two library preparation protocols and two sequencing platforms were applied to analyse six bacterial cultures and their corresponding DNA obtained as part of a proficiency test. The impact of each variable on sequencing results was assessed using the following parameters: reads quality, assembly and alignment statistics; number of single nucleotide polymorphisms (SNPs), detected applying assembly- and alignment-based strategies; antimicrobial resistance genes (ARGs), identified on de novo assemblies of all sequenced genomes. The investigated nucleic acid extraction procedures, library preparation kits and sequencing platforms do not significantly affect de novo assembly statistics and number of SNPs and ARGs. The only exception was observed for two duplicates, which were associated to one PCR-based library preparation kit. Results from this comparative study can support researchers in the choice toward the available pre-sequencing and sequencing options, and might suggest further comparisons to be performed.