Calcium peroxide (CaO2) nanoparticles have been extensively applied in treatment of contaminated groundwater through bioremediation or modified Fenton (MF) processes. In the present study utilization of CaO2 in bioremediation and MF (CaO2 +FeSO4) reaction is investigated for benzene (50 mg/L) removal in continuous flow sand-packed columns. The results indicated that MF produced OH radicals markedly increased benzene remediation at first 30 days (up to 93%). But, OH. generation rate was gradually declined when the pH was increased and finally 75% of initial benzene removed after 100d. In bioremediation column, because of supplying adequate oxygen by CaO2, the number of planktonic bacteria logarithmically increased to more than 5 x 10(6) CFU/mL (two orders of magnitude) and consequently 100% benzene removal was achieved by the end of experiment. Scanning electron microscopy analysis visualized the attached biofilm growth on sand surfaces in CaO2 injected columns indicating their key role in the remediation process. The impact of each process on the microbial biodiversity of groundwater was investigated by next generation sequencing (NGS) of the 16S rRNA gene. The alpha and beta analysis indicated that microbial diversity is decreased by CaO2 injection while benzene-degrading species such as Silanimonas, Arthrobacter and Pseudomonas spp. were dominated in re mediation column.