Cell-free circulating mitochondrial DNA (CFCmtDNA) is a damage-associated molecular pattern (DAMP) that can activate Toll-like receptor 9 (TLR-9). The main objectives of this case-control study were 1) to determine absolute concentrations and immunostimulatory capacity of CFCmtDNA, in membrane-bound and -unbound states, in cases with preeclampsia and healthy controls and 2) to implement a bootstrapped penalized regression analysis to establish the contribution of CFCmtDNA to preeclampsia diagnosis and its interaction with commonly collected patient characteristics. To determine the contribution of membrane-bound and -unbound CFCmtDNA in preeclampsia, DNA from plasma samples was exctracted with lysis buffer (membrane-unbound) and without lysis buffer (membrane-bound). CFCmtDNA, quantified using absolute PCR quantification protocol, was reduced in preeclampsia compared to healthy controls (P≤0.02). While the pattern of reduced CFCmtDNA in preeclampsia was similar between methods of DNA extraction, DNA isolation with membrane lysis buffer resulted in 1,000-fold higher CFCmtDNA quantification in the preeclampsia group (P=0.0014) and 430-fold higher CFCmtDNA quantification in the control group (P<0.0001). Even though CFCmtDNA concentrations were reduced, plasma from women with preeclampsia induced greater TLR-9 activation than plasma from gestational age matched controls (P≤0.01) as monitored using SEAP reporter 293 cells expressing human TLR-9. Penalized regression analysis showed that women with preeclampsia are strongly likely to have high concentrations of nDNA and DNase I along with a prior history of preeclampsia. Low concentrations of CFCmtDNA and mode of delivery were also associated with preeclampsia. In conclusion, our data demonstrate increases in the immunostimulatory potential of CFCmtDNA and upregulation of DNA degradation mechanisms in women with preeclampsia at the third trimester.