The aim is to optimize a rapid DNA extraction protocol for biosolids that provides high recovery and purity, and to evaluate potential biases on both quantitative and qualitative microbial analyses. A protocol with 3-step sequential DNA extractions using a modified lysis buffer and purification by a commercial DNA kit was developed and compared to a previously known high recovery solvent-based extraction method, a commercial soil DNA kit with single extraction, and the same commercial kit protocol except extracted with the modified lysis buffer. The developed protocol showed more than 7 times DNA recovery compared to the commercial kit, and comparable E. coli concentrations to the solvent-based method. Direct adoption of the commercial soil kit showed significantly lower recovery and underestimation of E. coli compared to the high recovery protocols. A simple switch of the lysis buffer also improved the DNA recovery by 5 times. Species diversity indexes from pyrosequencing analysis on the other hand showed scatter results from all protocols and only as much as 76% similarity was observed among any paired protocols. Phylogenetic analysis showed a shift of dominance as DNA recovery increases. Overall, this work shows that the developed protocol allows extraction of high quality and quantity DNA within 3 hr. However, qualitative comparisons of species structure may still vary.