Host defense peptides (HDP) constitute effector molecules of the innate immune system. Besides acting against microbia and fungi, they exhibit broad and selective oncolytic activity. The underlying mechanism is at least partially attributable to elevated surface-exposed levels of phosphatidylserine (PS) on tumor targets. In this study, comprehensive analysis of NK-2-based derivatives (C7A, C7A-D21K, and C7A-Δ) was done on patient-derived ultra-low passage colorectal carcinoma (CRC) cell lines. Peptides were designed to improve antitumoral potential. Mellitin was used as positive control and a non-toxic peptide (NK11) served as negative control. Subsequently, effectiveness of local HDP application was determined in xenopatients. Generally, CRC lines displayed a heterogeneous pattern of surface-exposed PS, which was usually below standard CRC cells. Of note, five out of seven cell lines were susceptible towards HDP-mediated lysis (lytic activity of peptides: C7A-D21K > C7A-Δ= C7A). Oncolytic activity correlated mostly with surface-exposed PS levels. Apoptosis as well as necrosis were involved in killing. In an in vivo experiment, substantial growth inhibition of HROC24 xenografts was observed after HDP therapy and, surprisingly, also after NK11 treatment. These promising data underline the high potential of HDPs for oncolytic therapies and may provide a rationale for optimizing preclinical treatment schedules based on NK-2.