Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden.