As potential in-situ chromate adsorbents for groundwater remediation, two different organo-bentonites having two structurally different surfactants with the same carbon chain length (hexadecyl trimethyl ammonium bromide (HDTMAB) and benzyl dimethyl hexadecyl ammonium chloride (BDMHDAC)) were prepared. The Fourier transformed infrared (FT-IR) spectroscopy, powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), and N2 adsorption-desorption isotherm characterization results of the prepared organoclays reveal that the HDTMAB and BDMHDAC moieties are mainly located on the interlayer and outer surface as HDTMAB-Bt (H-Bt) and BDMHDAC-Bt (B-Bt), respectively. Based on the chromate adsorption efficacy in terms of the amount of surfactant in organoclay, 1.5 times the cation exchange capacity of Bt was selected for further studies. The two different organoclays prepared herein exhibited fast chromate adsorption within 60 min and equilibrated at 240 min at 10 and 100 mg/L of chromate solution concentration. Based on the Langmuir and Freundlich isotherm analyses, H-Bt fitted well to the Langmuir isotherm, and B-Bt followed the Freundlich isotherm model owing to differences in the locations of the modified surfactants. In-situ chromate adsorption utilizing flow-through experiments were performed successfully under simulated soil conditions. The prepared B-Bt exhibited high chromate adsorption efficacy of around 81% at the adsorbent concentration of 3 g/300 mL within 170 min. Therefore, organoclays prepared using natural clay materials can be used as potential in-situ chromate adsorbents for groundwater remediation.