A multi-soil-layering system (MSL) is a biphasic soil-based wastewater treatment technology. In this study, two MSL columns with different soil mixture block (SMB) thicknesses were fed synthetic wastewater and monitored (i.e., organic matter and nutrients) to investigate the treatment performance of MSLs with horizontal flow (HF) orientation. The average removal efficiencies for System 1 (small SMBs) were 54%, 69%, 79%, 99%, and 95% for COD, TP, TN, NH3-N, and NO3--N, respectively, and 45%, 80%, 75%, 98%, and 85% for System 2 (large SMBs). The results suggest the primary function of SMBs in HF-MSLs is to provide phosphorous treatment. For nitrogen, unlike what has been found in other vertical flow (VF) MSL studies, denitrification was not a limiting factor. It is hypothesized (a) nitrification was facilitated by the permeable layer (PL, zeolite) and (b) the saturated conditions inherent to HF-MSLs promoted the growth of a heterogenous PL biofilm, proliferating denitrifying microorganisms. Based on the literature, it is theorized that organic matter (COD) removal was likely inhibited by the combination of competing bacterial species, insufficient aeration, a high influ-ent C/N ratio (12:1), and a relatively low HRT (~ 3 hrs). Overall, the bench scale removal efficiency results produced show the HF-MSL systems tested perform comparatively with VF-MSLs in addition to providing complete nitrogen removal. The findings from this intro-ductory investigation suggest HF-MSLs warrant further study. More research is needed (e.g., biological assessment) to validate the inter-pretations presented herein.